Maize plants with reduced gene silencing

ABSTRACT

Maize plants with reduced gene silencing are disclosed.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/034,802, filed Mar. 7, 2008, which is hereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No: CSREES-NRI-CGP-2005-35301-15891 awarded by the Department of Agriculture.

FIELD

The field generally relates to gene silencing in plants.

BACKGROUND

The goal of Zea mays L. (corn) breeding is to combine various desirable traits in a single variety/hybrid. Such desirable traits include greater yield, better stalks, better roots, resistance to pesticides, pests and disease, tolerance to heat and drought, reduced time to crop maturity, better agronomic quality, higher nutritional value, and uniformity in germination times, stand establishment, growth rate, maturity and fruit size.

Modern molecular biology and transgenic technologies (genetic engineering) have greatly accelerated the introduction of new genes and, hence, new traits into corn lines. While useful, genetic engineering is hampered by transgene silencing problems. Transgene silencing is a little understood process by which genes introduced by genetic engineering are silenced or turned off. As such, transgene silencing is a major impediment to the use of genetic engineering for corn improvement. At present, the solution to transgene silencing is to search through a large number of transgenic events for transgene loci that are active and stable. This is a painstaking and laborious process, which greatly increases the cost of corn breeding using genetic engineering techniques.

In addition to transgene silencing, there are other examples of gene silencing that are variable, unstable, but heritable. In corn these include the cycling of transposable elements between active and inactive states and paramutation, gene silencing that occurs through interactions between specific alleles of a gene. The mechanism of silencing is not understood in any case, but current hypotheses invoke heritable alterations to chromatin structure.

Although mutants are known in corn that can prevent or reverse gene silencing (U.S. Pat. No. 7,264,970), the molecular identity of these factors remains unknown. There thus remains a need to identify the molecular identity of these factors. There is also a need to reduce or mitigate gene silencing in transgenic plants and to reduce inbreeding depression during plant breeding.

SUMMARY

In order to meet these needs the present invention provides a method for reducing or mitigating gene silencing of a transgene by providing a transgenic plant with a transgene and introducing a recombinant nucleic acid to the transgenic plant, in which the recombinant nucleic acid includes a nucleic acid in an antisense orientation in which the sense orientation of the nucleic acid encodes a polypeptide selected from the group consisting of SEQ ID NOs 1-15, and in which the level of expression of the transgene is increased relative to the level of expression of the transgene in a transgenic plant that does not contain the recombinant nucleic acid.

The present invention is further directed to a method for reducing or mitigating gene silencing of a transgene by providing a transgenic plant with a transgene and introducing a recombinant nucleic acid to the transgenic plant, in which the recombinant nucleic acid includes an RNA interference (RNAi) construct including at least a fragment of 20 contiguous nucleotides of a nucleic acid that encodes a polypeptide including but not limited to SEQ ID NOs 1-15 and in which the level of expression of the transgene is increased relative to the level of expression of the transgene in a transgenic plant that does not contain the recombinant nucleic acid.

The methods may further include the step of screening the resulting plants for reduced gene silencing relative to the plant that does not contain the recombinant nucleic acid.

The present invention is further directed to a transgenic plant comprising a recombinant nucleic acid including a nucleic acid in an antisense orientation in which the sense orientation of the nucleic acid encodes a sequence selected from the group consisting of SEQ ID NOs 1-15.

The present invention is further directed to a transgenic plant comprising a recombinant nucleic acid including an RNA interference (RNAi) construct including at least a fragment of 20 contiguous nucleotides of a nucleic acid that encodes a polypeptide selected from the group consisting of SEQ ID NOs 1-15.

The present invention is further directed to seeds from the transgenic plants described above.

The present invention further describes a method for reducing or mitigating inbreeding depression in a plant by providing a plant and introducing a recombinant nucleic acid to the plant, in which the recombinant nucleic acid includes a nucleic acid in an antisense orientation in which the sense orientation of the nucleic acid encodes a polypeptide including but not limited to SEQ ID NOs 1-15, and in which the level of inbreeding depression is reduced relative to the level of inbreeding depression in a plant that does not contain the recombinant nucleic acid.

The present invention is further directed to a method for reducing or mitigating inbreeding depression in a plant by providing a plant and introducing a recombinant nucleic acid to the plant, in which the recombinant nucleic acid comprises an RNA interference (RNAi) construct including at least a fragment of 20 contiguous nucleotides of a nucleic acid that encodes a polypeptide selected from the group consisting of SEQ ID NOs 1-15, and in which the level of inbreeding depression is reduced relative to the level of inbreeding depression in a plant that does not contain the recombinant nucleic acid.

The methods may further include the step of screening the resulting plants for reduced inbreeding depression relative to the plant without the recombinant nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Comparison of pl1

Expression between rmr1 mutants and heterozygous siblings. pl1 RNA levels increase significantly in rmr1 mutants, while transcription rates of paramutant alleles are unaffected.

(A) The relative mean transcription rates from four independent sets of +/rmr1-3 (open) and rmr1-3/rmr1-3 (filled) siblings (±standard error of the mean) at the indicated loci.

(B) RNase protection analysis comparing pl1 and actin1 RNA levels in the same individual plants and tissues used for in vitro transcription analysis.

(C) Quantification of relative pl1 RNA levels from analyses as represented in (B).

FIG. 2: Map-Based Cloning of rmr1

(A and B) Rice Chromosome 5 (http://rice.tigr.org/) (A) and maize Chromosome 6 (B) (2005 FPC map, contig 285; http://www.genome.arizona.edu/fpc/maize/) with synteny of annotated rice loci and orthologous maize markers (gray boxes) highlighted. Black boxes indicate the rice rmr1 ortholog, Os05g32610, and the SSLP marker npi252. Neither rmr1 nor SSLP marker bnlg1174a are represented on the FPC map, though both can be amplified from a BAC (c0007N19), identified by GenBank ID AY109873, which maps to the region identified by the black line.

(C) Gene structure of rmr1 with exons in black; EMS-derived mutations are noted.

(D) Gray boxes highlight conserved Pfam SNF2_N (E-value=1.3×10⁻⁸; amino acids 851 to 1214) and Helicase_C (E-value=1.1×10⁻¹¹; amino acids 1255 to 1334) profiles in the RMR1 protein. Predicted translational consequences of each rmr1 mutation are indicated.

FIG. 3: RMR1 Defines a Monophyletic Clade Distinct from DRD1

Distance tree with bootstrap values produced from alignment (FIG. 8) of the predicted Snf2 domain with other Snf2 proteins: the tree shows that RMR1, CLSY1, and DRD1 (highlighted in gray) are members of a Rad54-like subfamily of Snf2 proteins. Three distinct monophyletic groups compose this subfamily, numbered 1 to 3. Prefixes: At, Arabidopsis; Os, rice; Sc, S. cerevisiae.

FIG. 4: Cytosine Methylation Patterns and Small RNA Accumulation Are Altered at Pl1-Rhoades in rmr1 Mutants

(A) Schematic of Pl1-Rhoades locus with exons highlighted in black and the upstream doppia element represented by the gray arrow. The methylation context of sites cut by methylation-sensitive enzymes are shown in parentheses. Open circles denote sites hypomethylated in rmr1-1 mutants while filled circles are sites methylated in both wild-type and rmr1 mutants. BsrI restriction sites and the regions used to generate probes for blot hybridization analysis, denoted A and B, are also shown.

(B and C) Representative Southern blots hybridized with probe A showing methylation status at a StuI site in rmr1 mutants and heterozygous siblings (B), as well as Pl′ and Pl-Rh plants (C) with a larger 2.9-kb band (upper arrow) representative of a fully methylated BsrI fragment, and a 2.1-kb band (lower arrow) indicative of a hypomethylated StuI site. Additional primary blots shown in FIGS. 9 and 14.

(D) Small RNA northern blot probed with doppia sequence from probe B showing changes in amount of small RNAs between rmr1-1 plants and wild-type (WT) siblings.

(E) Southern blot of genomic DNA digested with BstNI (“B” lanes) and methylation-sensitive PspGI (“P” lanes) hybridized with probe B, showing no bulk changes in doppia methylation genome-wide.

FIG. 5: Pl′ Establishment in an rmr1 Mutant Background

Plants with a T6-9 translocation chromosome carrying Pl1-Rhoades in the Pl-Rh state (dark anther pigmentation) and heterozygous for the rmr1-2 allele were crossed to Pl′ plants (light anther pigmentation) heterozygous for the rmr1-1 allele. Of the resultant progeny with semi-sterile pollen (heterozygous for the T6-9 interchange pair), plants homozygous for a mutation at rmr1 were chosen based on the dark anther phenotype. These plants were then crossed to a Pl-Rh tester with the expectation that in progeny inheriting the interchange, the expression status of the Pl1-Rhoades allele on the T6-9 translocation chromosome (T6-9 Pl(?)) would indicate if establishment of the Pl′ state was affected in the F1. The numbers represent the number of plants displaying a given anther phenotype, indicating that the Pl′ state was established on the interchange chromosome in the rmr1 mutants.

FIG. 6: Two General Models for RMR1 Action at the Pl1-Rhoades Allele

RMR1 maintains nonsymmetrical methylation of the doppia element (light gray arrow) upstream of the pl1 coding region (exons in black) via an RdDM pathway. Small RNAs are produced in a RMR1-dependent fashion with homology to the doppia element, and maize orthologs of characterized RdDM proteins, as well as RMR1, then act as effectors of these siRNAs, facilitating cytosine methylation at complementary sequences of the DNA template. In the model shown in (A), the heterochromatic region of doppia is maintained and established independently of the Pl1-Rhoades chromatin state, but derepression of the upstream repetitive element in an rmr1 mutant causes changes in the nearby genic region through processivity of RNA polymerase II or other general transcription factors that bind the upstream elements. In the model shown in (B), the doppia element is repressed by the same RdDM pathway shown in (A), but the Pl′ state represents a spread of the heterochromatic domain beyond the region targeted by the siRNAs for cytosine methylation. This spread might be mediated by RMR1 activity, or by another chromatin modifier. In (B), loss of RMR1 would lead to a loss of the repressive chromatin state at doppia and the ability for it to spread.

FIG. 7: Run-on transcription assay comparing rmr1 mutants to non-mutant heterozygotes.

Transcription rates of paramutant alleles are unaffected by Rmr1 action (A) In vitro radiolabeled RNAs corresponding to the indicated genes from isolated husk nuclei of sibling plants detected with slotblot hybridizations (pBS, bacterial plasmid DNA; pl1, purple plant1; b1, booster1; a1, anthocyaninless1; uq, ubiquitin). (B) Quantification of relative mean transcription rates from five independent sets of +/rmr1-1 (open) and rmr1-1/rmr1-1 (closed) siblings (±s.e.m.) showing no significant difference between pl1 transcription rates (n=5, 2-tailed 2-sample t-test, t=2.1, P=0.1). (C) In vitro radiolabeled RNAs from isolated husk nuclei of rmr1-3 mutants and heterozygous siblings used to generate quantification in FIG. 1.

FIG. 8: Alignment of RMR1 helicase domain with other known and predicted Snf2 proteins (SEQ ID NOs: 1-15)

FIG. 9: Southern blots comparing the DNA methylation status of the region upstream of Pl1-Rhoades in rmr1-1 mutants and heterozygous siblings.

Additional Southern blots used to generate methylation profile in FIG. 4A. (A) Genomic digests of an rmr1-1 mutant (−) and heterozygous sibling (+) using the listed methylation-sensitive restriction enzymes in concert with BsrI, hybridized with probe A (FIG. 4A). The top box defines the band representing the full length BsrI fragment, and lower boxes represent digest products hypomethylated at the restriction site being tested. A site was considered hypomethylated if the lower band was of greater intensity in the mutant as compared to the non-mutant and this was accompanied by a concomitant depletion of the upper band. (B) Blot hybridized with probe A comparing rmr1-1 mutants to heterozygous siblings with respect to methylation at a PspGI site. The top arrow represents a fully methylated BsrI fragment, the second arrow represents the expected size of a BsrI fragment hypomethylated at a PspGI site 123 bp upstream of Pl1-Rhoades, and the lowest arrow represents a hypomethylated PspGI site internal to doppia, 389 bp upstream.

FIG. 10: Comparison of Pl1-Rhoades upstream methylation status in rmr6-1 mutants and heterozygous siblings.

(A) Shows a methylation profile similar to that shown in FIG. 4A showing sites at the Pl1-Rhoades locus hypomethylated (open circle) in an rmr6-1 mutant as compared to heterozygous siblings. Blots (B) and (C) were used to generate this profile and are analogous to the blots shown for rmr1 mutants in FIGS. 10 A and B respectively.

FIG. 11: Comparison of Pl1-Rhoades upstream methylation status in mop1-1 mutants and heterozygous siblings.

(A) Shows a methylation profile similar to that shown in FIG. 4A showing sites at the Pl1-Rhoades locus hypomethylated (open circle) in a mop1-1 mutant as compared to heterozygous siblings. Blots (B) and (C) were used to generate this profile and are analogous to the blots shown for rmr1 mutants in FIGS. 10 A and B respectively.

FIG. 12: Additional small RNA northern blots showing accumulation of doppia small RNAs are lost in both sense and antisense orientations in rmr1 mutants.

Small RNA northern blots were probed with Probe B (FIG. 4A) in both the sense (A) and antisense (B) orientations showing small RNAs (˜26 nt) with doppia sequence similarity are present in sense and antisense orientation in rmr1-1 heterozygotes (+) and are lost in rmr1-1 mutants. 22 and 21 nt DNA oligos used as sizing standards are also shown.

FIG. 13: Mutations at rmr1 do not affect genome-wide methylation levels.

(A) Genomic DNA from four rmr1-3 mutants and non-mutant siblings, digested with BstNI (B) and a CNG methylation-sensitive enzyme, PspGI (P) which has the same recognition site, probed with radiolabled centromere sequence and (B) 45S repeat sequence. A comparison between the PspGI digests in mutant and non-mutant individuals reveals no gross methylation differences.

FIG. 14: Additional Southern blots showing no changes in Pl1-Rhoades methylation status between the Pl-Rh and Pl′ states.

(A) The methylation status of upstream PspGI sites was compared for Pl′/Pl′ and Pl-Rh/Pl-Rh plants with the Pl1-Rhoades allele introgressed into distinct A619 and A632 backgrounds via hybridization with probe A. The arrows are as indicated on FIG. 10B and the blot reveals no methylation differences at this site between the two Pl1-Rhoades regulatory states. The ‘C’ lanes indicated control lanes where the digest was carried out with the BstNI, methylation-insensitive, restriction enzyme.

(B) Is analogous to blot shown in FIG. 4C though the plants are from a different background (A619 introgression) than the plants used in FIG. 4C (A632 introgression) showing that there are no methylation differences at the StuI site in either background.

FIG. 15: RT-PCR expression profile of rmr1 in wild-type tissue S. Leaf, seedling leaf; A. Leaf, adult leaf; SAM, shoot apical meristem; Im. Tassel, immature tassel; Im. Ear, immature ear. RT-PCR was carried out using primers that span the first and second introns of rmr1.

FIG. 16: RMR1 and MOP1 small RNA profiles. Etbr staining of 20 μg enriched small RNAs following PAGE separation. Sizes of DNA oligonucleotides is noted for reference.

FIG. 17: Reactivation of silenced pWRWR transgenes.

(A) Schematic of pWRWR design.

(B) Pericarp phenotype of Pww null, Pww line with an active pWRWR transgene and subsequent silenced epigenotypes.

(C) Example of somatic reactivation of silenced pWRWR in ear of one rmr1-1 F2 homozygote and subsequent S1 progeny.

FIG. 18: pl1 paramutation.

Pl and Pl′ phenotypes from (A) seedling leaf sheath, (B) mature leaf sheath and (C) tassels.

(D) Quantification of pl1 expression in anther tissues is determined by a 1-7 graded series of Another Color Scores (ACS).

(E) Allelic behaviors. Pl is unstable and can, at various frequencies (indicated by dashed line), change to a transcriptionally repressed state referred to as Pl′ (spontaneous paramutation). Pl invariably (indicated by solid line) changes to a Pl′ state when Pl is exposed to Pl′ (induced paramutation). Pl′ can revert, at various frequencies (indicated by dashed line), to Pl, when heterozygous with some alleles or when hemizygous.

FIG. 19: RMR1 features.

(A) In silico translation of rmr1 genomic sequence highlighting a canonical bipartite nuclear localization signal (NLS, dark grey box, PredictNLS: http://cubic.bioc.columbia.edu/predictNLS/), highly conserved SNF2_N and Helic_C domains (black boxes), regions of high intrinsic disorder (underlined, see text), and locations of intended peptides for antisera production (white boxes). Position and nature of the rmr1-3 nonsense lesion is noted.

(B) RMR1 conserved domain (RMR1cd; aa813-1423) highlighting helicase motifs found within the SNF2_N and Helic_C profiles, and three non-functional RMR1 variants. Non-traditional motifs Ib, Ic, IVa, and Va were described by Dürr et al. (2006) in the analysis of the Sulfolobus solfataricus RAD54 structure; A and D denote motifs described by Thoma et al. (2005) in the analysis of D. rerio Rad54 structure that overlap with Ic and IVa. Functions of these motifs are based on available structural data: I—Walker A box, binds γ-P of ATP; Ia—3′ to 5′ DNA contacts; Ib—5′ to 3′ DNA contacts; Ic/C—minor grove contacts; II—Walker B box (DExx helicase motif), provides the Glu residue involved in ATP hydrolysis; Ia—thought to interface DNA binding to catalytic ATP hydrolysis by facilitating conformational change; III and VI—ATP binding; IV and V—DNA binding. Mutations in IVa inhibit ATP hydrolysis.

FIG. 20: Cell-autonomous transgene marker construct and root tips images from silenced and reactivated lines. (courtesy of J. Gutiérrez-Marcos)

DETAILED DESCRIPTION OF THE INVENTION

Genetics is founded on the principle that heritable changes in genes are caused by mutations and that the regulatory state of gene pairs (alleles) is passed on to progeny unchanged. An exception to this rule, paramutation, produces changes in gene control that are stably inherited without altering the DNA sequence. It is currently thought that these allelic interactions cause structural alterations to the chromatin surrounding the gene. Recent work in both maize and mice suggests that RNA molecules may be responsible for paramutations. Several genes are required to maintain the repressed paramutant state of a maize purple plant1 (pl1) allele, and here we report that one of these genes encodes a protein (RMR1) with similarity to a protein previously implicated in facilitating genomic DNA modifications via small RNA molecules. Genetic and molecular experiments support a similar role for RMR1 acting at a repeated sequence found adjacent to this pl1 gene. Although loss of these DNA modifications leads to heritable changes in gene regulation, the data indicate these changes do not represent the heritable feature responsible for paramutation. These findings highlight an unusual but dynamic role for repeated genomic features and small RNA molecules in affecting heritable genetic changes independent of the DNA template. Paramutations represent heritable epigenetic alterations that cause departures from Mendelian inheritance. While the mechanism responsible is largely unknown, recent results in both mouse and maize suggest paramutations are correlated with RNA molecules capable of affecting changes in gene expression patterns. In maize, multiple required to maintain repression (rmr) loci stabilize these paramutant states. Here we show rmr1 encodes a novel Snf2 protein that affects both small RNA accumulation and cytosine methylation of a proximal transposon fragment at the Pl1-Rhoades allele. However, these cytosine methylation differences do not define the various epigenetic states associated with paramutations. Pedigree analyses also show RMR1 does not mediate the allelic interactions that typically establish paramutations. Strikingly, our mutant analyses show that Pl1-Rhoades RNA transcript levels are altered independently of transcription rates, implicating a post-transcriptional level of RMR1 action. These results suggest the RNA component of maize paramutation maintains small heterochromatic-like domains that can affect, via the activity of a Snf2 protein, the stability of nascent transcripts from adjacent genes by way of a cotranscriptional repression process. These findings highlight a mechanism by which alleles of endogenous loci can acquire novel expression patterns that are meiotically transmissible.

The term “paramutation” describes a genetic behavior in which the regulatory state of specific alleles is heritably altered through interactions with their homologous partners in trans [1,2]. This behavior presents an exception to the Mendelian principle that alleles segregate from a heterozygous state unchanged [3]. Paramutations have been best characterized at loci encoding transcriptional regulators of pigment biosynthesis in maize, but similar behaviors have been described in other plant and animal systems, most recently in mice [4,5]. While the broader roles of paramutation in genome-wide regulation and evolution remain to be seen, the Pl1-Rhoades allele of the maize purple plant1 (pl1) locus presents a tractable system to study the paramutation process.

The pl1 locus encodes a Myb-like protein that acts as a transcriptional activator of genes required for anthocyanin pigment production [6]. Inheritance patterns illustrate that the Pl1-Rhoades allele can exist in quantitatively distinct regulatory states, reflected by differences in plant color. When individuals with a highly expressed reference state of Pl1-Rhoades, termed Pl-Rh, are crossed with plants having a repressed state, referred to as Pl′, only progeny with weak pigmentation are produced [7,8]. Pl-Rh states invariably change to Pl′ in Pl-Rh/Pl′ heterozygotes [7]; this is a typical hallmark of paramutation. Relative to Pl-Rh, the Pl′ state displays reductions in both Pl1-Rhoades RNA levels (˜10-fold) and transcription rate (˜3-fold) that are associated with a reduction in plant pigment [8]. This repressed Pl′ state is meiotically stable when maintained in a Pl1-Rhoades homozygote, with no reversion to Pl-Rh seen to date. Pl′ can, however, revert to Pl-Rh when heterozygous with some pl1 alleles other than Pl1-Rhoades, when maintained in a hemizygous condition, or in the presence of specific recessive mutations [9-12].

Genetic screens for ethane methyl sulfonate (EMS)-induced recessive mutations identify at least ten loci, including required to maintain repression1 (rmr1), rmr2, rmr6, and mediator of paramutation1 (mop1), whose normal functions maintain the repressed Pl′ state ([10,11,13]; J. B. H., unpublished data). These rmr mutations specifically affect the expression of Pl1-Rhoades and not other pl1 alleles [10,11], indicating that the Pl1-Rhoades allele is a direct and specific target of paramutation-based epigenetic changes. mop1 was recently identified [14,15] as encoding the putative ortholog of the Arabidopsis protein RDR2, a presumed RNA-dependent RNA polymerase involved in siRNA-based maintenance of de novo cytosine methylation [16]. Recessive mutations defining rmr1, rmr2, and rmr6 destabilize the repressed Pl′ state, resulting in darkly pigmented plant tissues, an increase in pl1 RNA levels, and meiotic transmission of Pl-Rh revertant states [10,11]. To date, the molecular identity of these rmr factors remains unknown. There thus remains a need to identify the molecular identity of these rmr factors. There is also a need to reduce or mitigate gene silencing in transgenic plants and to reduce inbreeding depression during plant breeding.

In this report we identify rmr1 as encoding a novel Snf2 protein that represents a founding member of a subgroup of factors similar to proteins involved in plant small RNA metabolism. Our analyses show that RMR1 affects both pl1 RNA transcript stability as well as small interfering RNA (siRNA) accumulation and DNA methylation patterns at Pl1-Rhoades. These results support a model in which maintenance of paramutant states is dependent on a repression mechanism similar to the recently proposed cotranscriptional gene silencing mechanism in fission yeast [17,18]. To our knowledge, RMR1 is the first protein identified that maintains trans-generationally repressed states established by paramutation.

DEFINITIONS

The term “plant” includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions and in most plant tissues. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

A “plant promoter” is a promoter capable of initiating transcription in plant cells.

An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. The expression vector can be an RNA or a DNA vector. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter (an expression cassette). An “expression cassette” refers to a subsequence of the expression vector.

The terms “substantially identical” or “substantial identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least about 60%, preferably 65%, 70%, 75%, preferably 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition, when the context indicates, also refers analogously to the complement of a sequence. Preferably, the substantial identity exists over a region that is at least about 6 7 amino acids or 25 nucleotides in length, or more preferably over a region that is 50 100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389 3402 (1977) and Altschul et al., J. Mol. Biol. 215:403 410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873 5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

Cloning of Target Nucleic Acids

Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning—A Laboratory Manual, (1989) or Current Protocols in Molecular Biology Volumes 1 3 (Ausubel, et al., eds. 1994 1998).

The isolation of nucleic acids corresponding to target genes may be accomplished by a number of techniques. For instance, oligonucleotide probes based on known sequences can be used to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatamers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as flowers, and a cDNA library which contains the target gene transcript is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which target genes or homologs are expressed.

The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned target gene. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against an target polypeptide can be used to screen an mRNA expression library.

Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the target genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis et al., eds. 1990).

Polynucleotides may also be synthesized by well-known techniques as described in the technical literature (see, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411 418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Promoters and Expression Vectors

As described below, a vector containing a recombinant nucleic acid can be introduced into a plant by any suitable method. For example, the construct can be introduced into a plant via stable transformation with Agrobacterium, particle bombardment, electroporation, or transduction with a viral particle. A suitable expression vector is therefore selected according to the desired method of plant transformation.

In one embodiment, the construct is expressed via a DNA expression vector. Such expression vectors comprise DNA dependent RNA polymerase promoters that are active in plant cells, e.g., constitutive plant promoters such as those described herein and above (e.g., the nopaline synthase promoter, Sanders et al., Nuc. Acids Res. 15:1543 1558 (1987); or the CaMV 35S promoter, Urwin et al., Mol. Plant Microbe Interact. 10:394 400 (1997)) or tissue specific plant promoters such as those described herein and above.

In another embodiment, the gene silencing construct is transcribed via an RNA expression vector. The RNA expression vector encodes an RNA dependent RNA polymerase active in plant cells, and the gene silencing construct is transcribed via an RNA dependent RNA polymerase promoter active in plant cells. Suitable RNA dependent RNA polymerases and their corresponding promoters and expression vectors are derived, e.g., from potato virus X (Chapman et al., Plant J. 2:549 557 (1992), tobacco mosaic virus (see, e.g., Dawson et al., Virology 172:285 292 (1989)), tobacco etch virus (see, e.g., Dolja et al., Proc. Nat'l Acad. Sci. USA 89:10208 10212 (1992)), tobacco rattle virus (see, e.g., Ziegler-Graff et al., Virology 182:145 155 (1991)), tomato bushy stunt virus (see, e.g., Scholthof et al., Mol. Plant Microbe Interact. 6:309 322 (1993)), brome mosaic virus (see, e.g., Mori et al., J. Gen. Virol. 74:1255 1260 (1993)). Such expression vectors are prepared using techniques known to those of skill in the art, e.g., by using bacterial RNA polymerases such as SP6 and T7 followed by manual inoculation, or by introduction of the vectors into plants by Agrobacterium-mediated transformation (Angell & Baulcombe, EMBO J. 16: 3675 3684 (1997)).

In another embodiment, optionally, a DNA expression vector also comprises a gene encoding an RNA dependent RNA polymerase active in plant cells. The RNA dependent RNA polymerase is then used to amplify the construct (either the positive and/or the negative strand).

In another embodiment, the construct is expressed via a DNA expression vector derived from a plant DNA virus, e.g., cauliflower mosaic virus (see, e.g., Futterer & Hohn, EMBO J. 10:3887 3896 (1991), African cassaya mosaic virus (see, e.g., Ward et al., EMBO J. 7:1583 1587 (1988)) and the tomato golden mosaic virus.

In the present invention, a plant promoter may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Such genes include for example, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125 139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196 203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167 1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al., J. Mol. Biol. 208:551 565 (1989)), and Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol Biol. 33:97 112 (1997)).

Alternatively, the plant promoter may direct expression of the construct in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control (i.e. inducible promoters). Examples of environmental conditions that may effect transcription by inducible promoters include pathogen challenge, anaerobic conditions, elevated temperature, the presence of light, or spraying with chemicals/hormones. One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.

A number of tissue-specific promoters can also be used in the invention. For instance, promoters that direct expression of nucleic acids in roots and feeding cells can be used. In particular, such promoters are useful for using the methods of the invention to inhibit nematode endoparasites that live in roots. The root-specific ANR1 promoter is suitable for use in the present invention (Zhang & Forde, Science 279:407 (1998)). The wound specific promoter wun-1 from potato can be used, as it respond to intracellular root migration by Globodera sp. (see, e.g., Hansen et al., Physiol. Mol. Plant Pathol. 48:161 170 (1996)). Other genes that demonstrate parasitic nematode feeding-cell specific expression have been reported, and their promoters are suitable for use in the present invention (see, e.g., Bird et al., Mol. Plant Microbe Interact. 7:419 424 (1994); Gurr et al., Mol. Gen. Genet. 226:361 366 (1991)); Lambert et al., Nucl. Acids. Res. 21:775 776 (1993); Opperman et al., Science 263:221 223 (1994); Van der Eycken et al., Plant J. 9:45 54 (1996); and Wilson et al., Phytopathology 84:299 303 (1992)). Phloem specific promoters, which can be used to express the gene silencing construct of the invention for uptake by sap-sucking insects, include those referenced in Shi et al., J. Exp. Bot. 45:623 631 (1994).

The vector comprising the gene silencing construct will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfaron or Basta.

Plant Transformation

Expression vectors of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the expression vector may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the expression vectors can be introduced directly to plant tissue using ballistic methods, such as particle bombardment. In addition, the constructs of the invention may be introduced in plant cells as DNA or RNA expression vectors or viral particles that co-express an RNA dependent RNA polymerase.

Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of expression vectors using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717 2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70 73 (1987).

Alternatively, the expression vectors may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens—mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature (see, e.g., Horsch et al., Science 233:496 498 (1984); Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants (Potrykus, ed. 1995)).

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as enhanced resistance to pathogens. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124 176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21 73 (1985). Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of plant Phys. 38:467 486 (1987).

The nucleic acids of the invention can be used to confer desired traits on essentially any plant. Thus, the invention has use over a broad range of plants, including wheat, corn, rice, sorghum, pepper, tomato, squash, banana, strawberry, carrot, bean, cabbage, beet, cotton, grape, pea, pineapple, potato, soybean, yam, and alfalfa, as well as other species described herein.

One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants, if such a technique is used, and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

Using known procedures one of skill can screen for plants of the invention by detecting the effect of the construct of the invention in the target organism, either using in vitro assays such as plant culture, or in vivo assays such as transgenic plants. Means for directly and indirectly detecting and quantitating protein and RNA expression in vitro and in cells are well known in the art.

Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well established techniques as described above. Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homolog, of the invention, standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.

The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al., Eds., (1984) Handbook of Plant Cell Culture—Crop Species, Macmillan Publ. Co., New York, N.Y.; Shimamoto et al. (1989) Nature 338: 274-276; Fromm et al. (1990) Bio/Technol. 8: 833-839; and Vasil et al. (1990) Bio/Technol. 8: 429-434.

Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.

Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.

After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

Antisense and RNAi Constructs

Antisense and RNAi treatments represent one way of altering rmr1 expression in accordance with the invention. In particular, constructs comprising an rmr1 sequence, including fragments thereof, in sense and/or antisense orientation, may be used to decrease or effectively eliminate its expression in a plant.

Techniques for RNAi are well known in the art and are described in, for example, Lehner et al., (2004) and Downward (2004). The technique is based on the fact that double stranded RNA is capable of directing the degradation of messenger RNA with sequence complementary to one or the other strand (Fire et al., 1998). Therefore, by expression of a particular coding sequence in sense and antisense orientation, either as a fragment or longer portion of the corresponding coding sequence, the expression of that coding sequence can be down-regulated.

Antisense methodology takes advantage of the fact that nucleic acids tend to pair with complementary sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host plant or part thereof. In certain embodiments of the invention, such an RNAi or antisense oligonucleotide may comprise any unique portion of a nucleic acid sequence provided herein.

Constructs may be designed that are complementary to all or part of the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes a construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an RNAi or antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see above) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES Example 1 Rmr1 Defects Affect Pl1 RNA Stability

The rmr1 locus is defined by four recessive mutations characterized by a darkly pigmented plant phenotype that results from loss of Pl repression. Previous RNase protection experiments showed a 26-fold increase in pl1 RNA in floret tissue between rmr1-1 mutant plants and heterozygous siblings [10]. However, these experiments did not address if changes in pl1 transcript abundance correlated with changes in actual transcription at the pl1 locus.

In vitro transcription assays using nuclei isolated from husk leaf tissue revealed there was no statistically significant change in relative transcription rates of the Pl1-Rhoades allele between rmr1-1 mutants and heterozygous siblings (FIG. 7). However, transcription rates of anthocyaninless1 (a1), a direct target of the PL1 transcriptional activator [7,19], were ˜4-fold greater in rmr1-1 mutants (FIG. 7), reflecting significantly increased PL1 activity. Transcription rates from colored plant1 (b1)—a locus encoding a basic helix-loop-helix factor genetically required for a1 transcription—remained unchanged. These results were recapitulated in comparisons between nuclei isolated from rmr1-3 mutants and heterozygous siblings in which in vitro transcription assays revealed no significant change in transcription rate of Pl1-Rhoades (FIGS. 1A and 7; n=4, two-tailed two-sample t-test, t=0.8, p=0.5) while RNase protection experiments showed a 5.7-fold increase in pl1 RNA for rmr1-3 mutants (FIGS. 1B and 1C; n=2, two-tailed two-sample t-test, t=10.8, p<0.01) using RNA isolated from the same tissues of the same individuals. Similar comparisons from identical tissues but in a different genetic background again showed that transcription rates at pl1 remained unchanged while pl1 RNA levels increased 7.52-fold in rmr1-3 mutants compared to heterozygous siblings (n=1; see).

These RNA expression results sharply contrast those of previous reports using identical in vitro transcription assays that detected significant differences in Pl1-Rhoades transcription rates between Pl′ and Pl-Rh states and between rmr6 mutants and non-mutants [8,11]. This indicates our in vitro results represent an accurate assessment of transcription rates and not a limitation of the assay to detect rate differences at the pl1 locus. Combined, these results imply an increase of pl1 RNA abundance disproportionate to insignificant changes in transcription rate in rmr1 mutants, the most direct interpretation being that RMR1 functions at a post-transcriptional level to stabilize Pl1-Rhoades RNA.

Example 2 Rmr1 Encodes a Novel Protein with a Snf2 Domain

To better understand rmr1 function and the paramutation mechanism, we used a map-based approach to identify the rmr1 gene. Using a polymorphic F2 population we looked for genetic linkage between the mutant phenotype and previously mapped chromosome markers [20]. The dark-color phenotype of rmr1-1 homozygotes showed invariant cosegregation with the mutant parent polymorphism of SSLP markers bnlg174a (680 chromosomes tested; <0.15 cM) and npi252 (60 chromosomes tested; <1.7 cM), indicating rmr1 was tightly linked to those markers in bin 6.05 on Chromosome 6. We used the high degree of synteny between this region and rice Chromosome 5 to identify candidate rmr1 orthologs (FIGS. 2A and 2B).

Within the syntenic rice region we identified a gene model, Os05g32610 (http://rice.tigr.org/), predicted to encode a Snf2 protein. The Snf2 protein family is composed of members similar to Saccharomyces cerevisiae Snf2p with a bipartite helicase domain containing Pfam SNF2_N and Helicase_C profiles, and includes many proteins involved in ATP-dependent chromatin remodeling [21,22]. While there was no public maize expressed sequence tag for this candidate, we used BLAST searches to identify genomic survey sequence similar to Os05g32610. Oligonucleotide primers were designed from these sequences and used to generate PCR amplicons spanning the maize Os05g32610 ortholog, which were sequenced from individuals homozygous for rmr1 progenitor alleles and mutant derivatives (see Example 5). The maize sequence generated from each of the homozygous mutants revealed single unique transition-type base pair changes consistent with EMS mutagenesis relative to the progenitor (FIG. 2C). The amino acid change associated with the rmr1-1 allele is predicted to prevent proper folding of the helicase domain [23], while the non-conservative amino acid substitutions associated with the rmr1-2 and rmr1-4 alleles occur at highly conserved residues in the SNF2_N profile (FIG. 2D). The rmr1-3 allele is associated with a nonsense mutation predicted to truncate the peptide before the conserved helicase domain (Full rmr1 sequence information is disclosed in GenBank accession numbers EU154999, EU155000, EU155001, EU155002, EU155003, EU155004, and EU155005; SEQ ID NOs: 16-29). CAPS markers were designed to the potential rmr1-1 and rmr1-3 lesions and used to show that the base pair polymorphisms at each of the probable lesions invariably cosegregate with the mutant phenotype (see Example 5). These results support these polymorphisms as bona fide molecular lesions in the rmr1 gene. Based upon molecular genetic mapping data, DNA sequencing results, and the relevance of the fact that Snf2 proteins affect chromatin environments, we conclude the rmr1 locus encodes a protein containing a Snf2 helicase domain.

Os05g32610 gene models and our cDNA sequencing analysis (see Example 5) indicate rmr1 encodes a 1,435-amino-acid protein. In addition to having the conserved Snf2 helicase domain, the protein has a large N-terminal region with no significant identity to any known or predicted proteins. Phylogenetic comparison with other known Snf2 proteins in maize, rice, Arabidopsis, and budding yeast shows RMR1 is a member of a Rad54-like subfamily defined by DRD1 (FIG. 3). Arabidopsis DRD1 is a putative chromatin remodeling factor affecting RNA-directed DNA methylation (RdDM) patterns [24-26]. In the emerging RdDM pathway model, DNA sequences are targeted for de novo cytosine methylation by complementary siRNA molecules generated from “aberrant” RNA transcripts. The putative MOP1 ortholog in Arabidopsis, RDR2, is required in this pathway to presumably generate double-stranded RNA from these transcripts and provide a substrate for siRNA biogenesis through activity of a Dicer-like enzyme [27]. DRD1 is thought to be a downstream effector protein that facilitates de novo methylation of targeted DNA sequences, possibly by modulating chromatin architecture to provide access to de novo methyltransferases [24-26,28]. The DRD1 subfamily also includes the recently identified CLSY1 protein implicated in the systemic spreading of siRNA-mediated silencing in Arabidopsis [29].

Multiple sequence alignments (FIG. 8) indicate RMR1 is not the structural ortholog of either DRD1 or CLSY1. The DRD1 subfamily can be divided into three distinct monophyletic groups, with RMR1, DRD1, and CLSY1 defining different groups (FIG. 3). The presumed maize ortholog of DRD1 is likely one of two proteins in the DRD1 subgroup, Chromatin remodeling complex subunit R 127 (CHR127) (http://chromdb.org/), a partial protein predicted from maize expressed sequence tag sequences, or CHR156, a full-length protein predicted from maize genomic sequence (see Example 5). RMR1 is more similar to Arabidopsis proteins predicted from At1g05490 and At3g24340. RNA interference knockdowns of these putative Arabidopsis orthologs are known to have little to no effect in response to DNA damage [30].

Taking into account the phylogenetic analysis of the predicted coding sequence, it is possible RMR1 function may be similar to, but distinct from, that of DRD1 and CLSY1. The three proteins may fulfill a similar role in RdDM, but perhaps function under different conditions or in distinct genomic contexts. Alternatively, they could perform different roles within an RdDM pathway, or function in separate epigenetic mechanisms altogether. Given the results of our pl1 RNA expression analyses, it is possible that RMR1 represents a Snf2 protein that links chromatin organization to RNA transcript stability.

Example 3 RMR1 Maintains Cytosine Methylation and Small RNA Accumulation at Pl1-Rhoades

In the described Arabidopsis RdDM pathway, DRD1 maintains cytosine methylation at nonsymmetrical CNN sequences represented by siRNAs [24-26]. Many endogenous genomic targets of DRD1 appear to be repetitive elements [31]. At Pl1-Rhoades there is a 402-bp terminal fragment of a CACTA-like type II DNA transposon, similar to doppia, 129 bp upstream of the translational start site [8,32,33]. Assuming analogous functional roles of RMR1 and DRD1 we compared DNA methylation patterns at this upstream repetitive element in rmr1 mutants and non-mutant siblings.

Previous restriction-enzyme-based comparisons of DNA methylation status between Pl-Rh and Pl′ states found no differences, although few 5′ proximal sites were evaluated [8]. Using Southern blot hybridization analysis following digestion of genomic DNA with methylation-sensitive restriction enzymes, we found that the doppia fragment is hypomethylated at specific sites in plants homozygous for the rmr1-1 mutation compared to heterozygous wild-type siblings (FIGS. 4A, 4B, and 9). Consistent with findings in Arabidopsis RdDM mutants [16, 34-36], the sites hypomethylated in rmr1 mutants were of the CNN context. A relative hypomethylation pattern in 5′ sequences is also present in plants homozygous for mutations at either rmr6 or mop1 (FIGS. 10 and 11). In rmr6 mutants the extent of hypomethylation was greater than that of either rmr1 or mop1 mutants and encompassed CG methylation sites as well as non-CG targets, suggesting rmr6 has a broader effect in cytosine methylation maintenance. The presence of these methylation differences in multiple mutant backgrounds indicates that this hypomethylation pattern reflects the chromatin status at doppia in plants where maintenance of repressed paramutant states is compromised.

Consistent with the Arabidopsis RdDM model, small RNAs (˜26 nt) with sequence similarity to the doppia element are detected in wild-type Pl′ plants in both sense and antisense orientations (FIGS. 4D and 12). These small RNAs are undetectable in rmr1 mutants, unlike in wild-type siblings. This result contrasts those in Arabidopsis showing that DRD1 deficiencies do not affect the abundance of endogenous siRNAs representing repetitive elements [31]. However, it has been reported that the abundance of endogenous siRNA and trans-acting siRNA populations are highly reduced in CLSY1 mutants [29].

To test if the doppia fragment hypomethylation was indicative of genome-wide changes we assayed the cytosine methylation status at centromeres and 45S repeat sequences. Cytosine methylation patterns were unaffected in either of these regions in rmr1 mutants as compared to non-mutant siblings (FIG. 13). Additionally, we examined the methylation status of doppia-like loci genome-wide (FIG. 4E) and found no obvious differences between rmr1 mutants and non-mutant siblings. These results indicate that while RMR1 acts on the doppia sequence upstream of Pl1-Rhoades, doppia elements appear unaffected throughout the genome. This specificity of RMR1 function may be due to its intimate and exclusive involvement with alleles that undergo paramutation, or may be indicative of differential regulation of repetitive elements depending on their genomic and epigenetic context.

If RMR1 is involved in maintaining cytosine methylation patterns characteristic of repressed paramutant states then a prediction would be that the methylation differences seen between mutants and non-mutants would reflect the Pl′ and Pl-Rh regulatory states. Surprisingly, there are no methylation differences at the doppia fragment between Pl-Rh and Pl′ states (FIGS. 4C and 14). These results suggest that while the upstream doppia element of Pl1-Rhoades is a target of multiple factors involved in maintaining the epigenetic repression associated with paramutation, the actual process of paramutation does not result in similar changes of DNA methylation at this element.

Example 4 RMR1 Is Not Required for Establishment of Paramutant States

Based on a reverse transcriptase PCR (RT-PCR) expression profile (FIG. 15) rmr1 appears to be expressed in all rapidly dividing somatic tissues, consistent with a role in maintaining paramutant states throughout development. However, since the methylation patterns maintained by RMR1 appear unrelated to the paramutant state of Pl1-Rhoades, we questioned whether RMR1 is directly required for paramutation to occur. This process results in the invariable establishment of the Pl′ state in Pl′/Pl-Rh plants, as evidenced by the observation that only Pl′/Pl′ progeny are found when Pl′/Pl-Rh plants are crossed to Pl-Rh/Pl-Rh testers [7,8]. If RMR1 were directly involved in this process we would expect that an rmr1 deficiency might interfere with the Pl′ establishment event. To test this, we tracked the behavior of individual Pl1-Rhoades alleles in test crosses to assess the ability of the Pl′ state to facilitate paramutations in Pl′/Pl-Rh; rmr1-1/rmr1-2 plants. The Pl1-Rhoades allele in a Pl-Rh state was genetically linked (˜1.5 cM) to a T6-9 translocation breakpoint. The T6-9 interchange can act as a dominant semi-sterility marker, allowing us to trace specific Pl1-Rhoades alleles through genetic crosses [11]. rmr1 mutants heterozygous for the T6-9 interchange (T6-9 Pl-Rh/Pl′) were crossed to a Pl-Rh/Pl-Rh tester (FIG. 5; Table 1). If establishment of the Pl′ state was prevented in rmr1 mutants, we would expect all progeny receiving the interchange to display a Pl-Rh/Pl-Rh phenotype (dark anther pigmentation). We observed that over half the progeny inheriting the interchange displayed a Pl′/Pl′-like phenotype (light anther pigmentation), indicating that paramutation was established in the rmr1 mutant parent. It should also be noted that Pl-Rh/Pl-Rh plants, and those of an intermediate phenotype of partial pigmentation [7], were present in both progeny inheriting the interchange and those inheriting a normal chromosome. These results are consistent with previous work showing Pl′ can revert to a Pl-Rh state in rmr1 mutants [10].

Corresponding analysis of the establishment of paramutant states at the b1 locus generated similar results (Table 2). The repressed B′ state of the B1-Intense allele [37] was established in B′/B-I rmr1 mutants greater than 95% of the time. While it is possible that rmr1 defects affect establishment efficiency, it will be difficult to differentiate any such effects from its clear role in maintenance [11]. These results point to an interesting duality in RMR1 function in which the wild-type protein is necessary for meiotic heritability of repressed epigenetic states, but is not required to establish these states. This duality is markedly different from results generated in the analysis of DRD1, which was shown to be necessary for the maintenance, establishment, and removal of repressive epigenetic marks [24,25].

Example 5 Materials and Methods

Scoring of the Pl1-Rhoades Allele Expression State and Rmr Mutants

Plants were scored as carrying Pl-Rh or Pl′ states through visual inspection of anther pigmentation and assignment of an anther color score as previously described [7]. Pl′/Pl′ (anther color score 1 to 4) anthers show little to no pigmentation while Pl-Rh/Pl-Rh (anther color score 7) anthers are dark red to purple. Mutants were scored in the same way, with rmr and mop mutants showing a Pl-Rh/Pl-Rh-like phenotype, except in the case of the F2 rmr1 mapping populations, in which mutants were chosen on the basis of a dark seedling leaf phenotype [10].

Genetic Stocks

Elite inbred lines (B73, A619, and A632) were provided by the North Central Regional Plant Introduction Station (http://www.ars.usda.gov/main/site_main.htm?modecode=36-25-12-00). Color-converted versions of A619 and A632 inbred lines were created by introgressing the Pl1-Rhoades allele into each [11]. The rmr1-1, rmr1-2, mop1-1, and rmr6-1 alleles have been previously described [8,10,13]. The rmr1-3 allele was derived from identical materials used to isolate rmr1-1 and rmr1-2; rmr1-4 was derived from EMS-treated pollen from an A619 color-converted line applied to a color-converted A632 line [11] (see Table 3 for complementation tests). The T6-9 translocation line carrying the Pl1-Rhoades allele used in Pl′ establishment tests has been described previously [11].

Pl1 Expression Analyses

In vitro transcription assays (rmr1-1 and rmr1-3; FIGS. 1 and 7) and RNase protection assays (rmr1-3 only; FIG. 1) were carried out as described [8] with husk nuclei and RNA isolated from single ears of the same genetic stocks used to measure pl1 RNA differences in rmr1-1 anthers [10]. The b1 and pl1 genotypes of these plants are as follows: B-Intense (B-I)/B-I, Pl1-Rhoades (Pl′) rmr1/Pl′ rmr1-1 and B-I/B-I; Pl′ rmr1-1/Pl′ rmr1-1, or B-I/B-I, Pl′ rmr1/Pl′ rmr1-3 and B-I/B-I; Pl′ rmr1-3/Pl′ rmr1-3. Identical procedures were applied to single ears from plants homozygous for Pl′ and either homozygous or heterozygous for rmr1-3 following a single backcross into the KYS inbred line [12].

Genetic Mapping of Rmr1

A F2 mapping population was created from inbred (S9) rmr1-1/rmr1-1, Pl′/Pl′, and color-converted A632 inbred (Pl′/Pl′, >93% A632) parents. DNA was isolated using the DNeasy 96 plant kit (Qiagen, http://wwwl.qiagen.com/) from F2 mutant seedlings, mapping parents, and F1 hybrid leaf tissue. These DNA samples were screened with SSLP markers developed from the Maize Mapping Project (http://www.maizemap.org/; US National Science Foundation award number 9872655; primer sequences and protocol available at http://maizegdb.org/). Initial marker choice was restricted to Chromosomes 6 and 9 because of linkage of rmr1 to a T6-9 breakpoint. In addition to the rmr1-1 mapping population, a second F2 mapping population created with inbred (S7) rmr1-3/rmr1-3, Pl′/Pl′, and color-converted A632 parents showed similar cosegregation with marker bnlg1174a (178 chromosomes tested; <0.56 cM). CAPS [61] markers were designed to test cosegregation of the rmr1-1- and rmr1-3-associated lesions with the rmr1 mutant phenotype. No recombinant chromosomes (876 chromosomes tested for rmr1-1,268 chromosomes tested for rmr1-3) were found using either marker.

Candidate Gene Selection and Sequencing

A BLAST search using the rice Os05g326100RF as a query identified maize GSS and sorghum expressed sequence tag sequences that were used to generate a contig representing the putative maize gene (see Candidate Gene Selection and Sequencing section below for sequence identifiers). Oligonucleotide primers (Sigma-Genosys, http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html) were designed from these sequences and used in PCR amplification of genomic DNA from three separate individuals homozygous for each rmr1 mutant allele as well as functional reference alleles rmr1-B73, rmr1-A632, and rmr1-A619. PCR amplicons were purified using QIAquick gel extraction kit (Qiagen) and dideoxy sequenced (UC Berkeley DNA Sequencing Facility, http://mcb.berkeley.edu/barker/dnaseq/). To verify the intron/exon structure of rmr1, cDNA was generated from rmr1-1 mutants as well as non-mutant B73 plants as described [15], and rmr1 was amplified via RT-PCR. The resulting products, which were the predicted size for spliced rmr1 transcript, were sequenced to validate the intron/exon structure shown in FIG. 2. See Table 4 for oligonucleotide primer sequences used (SEQ ID NOs: 30-46).

Phylogenetic Analysis

Sequencing reads from genomic and cDNA were aligned and edited with Sequencer (Gene Codes, http://www.genecodes.com/) to create a contig representing rmr1. The N-terminal prediction is based on alignment of RMR1 with the protein model for Os05g32610. A search of the Pfam database (http://www.sanger.ac.uk/Software/Pfam/) with the predicted RMR1 protein sequence was used to identify the conserved SNF2_N and Helicase_C protein profiles of the Snf2 helicase domain. MUSCLE [62] was used to generate an alignment between RMR1 and proteins from Arabidopsis, rice, maize (CHR127 and CHR156), and budding yeast over the helicase domain (FIG. S2). Sequences for CHR127 and CHR156 were retrieved from ChromDB (http://www.chromdb.org/). Additional sequence information for CHR156 was identified from BAC CH201-3L17 (GenBank accession AC194602), and gene model prediction was performed using FGENESH+ (Softberry, http://www.softberry.com/) with RMR1 as similar protein support. A distance tree was created and bootstrap values were calculated using PAUP* 4.0 from the above alignment (Sinauer Associates, http://www.sinauer.com/).

Southern Blot Analysis

Genomic DNA was isolated as described [63] from the terminal flag leaves of adult plants segregating for rmr1, rmr6, and mop1 mutants and heterozygous siblings as well as Pl′ and Pl-Rh plants as assayed by anther pigmentation [7,8,10,13]. Restriction digest and subsequent Southern blots were carried out as previously described [13], using the restriction enzymes listed in FIG. 4 (New England Biolabs, http://www.neb.com/). The probes specific to pl1 are shown in FIG. 4; the 45S and centromere probes are as described [13].

Small RNA Northern Blots

Small RNAs were prepared from 10-mm immature ear tissue and used to generate small RNA northern blots as previously described [64]. In FIG. 4D the small RNAs were run with a 27-bp DNA oligonucleotide containing doppia sequence that hybridized with the riboprobe used to identify the small RNAs. The riboprobe was synthesized as described [64] from a plasmid containing the region denoted probe B in FIG. 4A linearized at an AseI site so as to contain only doppia sequence.

Pl′ Establishment Tests

Establishment of the Pl′ state in rmr1 mutants was assayed essentially as described previously [11]. When the T6-9 interchange pair is heterozygous with structurally normal chromosomes, the plants display ˜50% pollen sterility due to meiotic-segregation-induced aneuploidy in the resulting gametes. Pollen sterility was assayed in the field using a pocket microscope. rmr1 mutants were crossed to Pl-Rh/Pl-Rh A619 or A632 inbreds (Table 1), and the resultant progeny were scored with respect to Pl1-Rhoades expression state.

Complementation Tests of New Mutant Alleles

Complementation test results (Table 3) show ems98292 and ems98941 mutations define alleles of mop1 and that ems98287 and ems051069 mutations define alleles of the rmr1 locus. The ems98287, ems98292, and ems98291 mutations were isolated using an ems pollen mutagenesis and genetic screening strategy as previously described. The ems051069 mutation was similarly generated and isolated using color converted A619 and A632 inbred parental lines.

Homozygous ems98262/ems98262 plants having a Pl-Rh-like phenotype (ACS 7) were outcrossed to Pl′/Pl′ testers. All F1 progeny plants (39 individuals from 3 independent outcrosses) had a Pl′-like anther phenotype (5 ACS 1; 22 ACS 2; 12 ACS 3) indicating the ems98262 allele is recessive and further that Pl′ alleles transmitted from homozygous ems98262/ems98262 plants are capable of inciting paramutation. The Pl-Rh-like anther phenotype was recovered in 10/62 F2 plants derived from three independent self-pollinations of F1 plants (30 ACS 1; 15 ACS 2; 2 ACS 3; 10 ACS 7). The observed frequency of Pl-Rh-like phenotypes (16%) is not significantly different from the 25% expected from a single locus recessive mutation (Pearson's goodness of fit x2=0.28; P=0.59). All F2 progeny with ACS 7 anthers were ˜⅔ the height of their Pl′-like siblings. Five of the 62 F2 progeny produced barren (no flowers) tassels so it was not possible to assign these to a given Another Color Score class. However, these five anther-less progeny were short in stature and had very strong plant color similar to all other F2 plants that had ACS 7 anthers. If we include these five anther-less progeny among the “ACS 7” mutant class, then 15/62 (24%) of the F2 progeny have the mutant phenotype. These F2 segregation ratios are consistent with the hypothesis that the dark-anther and dark-plant phenotypes are due to a single locus recessive mutation. Homozygous ems98941/ems98941 plants having a Pl-Rh-like phenotype were outcrossed to Pl′/Pl′ testers. All F1 progeny plants (14 individuals from 2 independent outcrosses) had a Pl′-like anther phenotype (2 ACS 1; 9 ACS 2; 2 ACS 3; 1 ACS 4) indicating that the ems98941 allele is recessive and further that Pl′ alleles transmitted from homozygous ems98941/ems98941 plants are not recalcitrant to subsequent paramutation.

Results of genetic crosses (Table 3) indicate the ems98262 and ems98941 mutations complement mutations at rmr1, rmr2 and rmr6. However, the two mutations fail to complement each other and the mop1-1 mutation. If the ems98262 mutation represents an allele of mop1, then ½ of all progeny (14-15 individuals) from the two complementation crosses with mop1-1 should have a Pl-Rh-like anther phenotype. The observed frequency of Pl-Rh-like types is not significantly different from the expected frequency for this hypothesis (Pearson's goodness of fit x2=3.07; P=0.08) suggesting ems98262 defines an allele of mop1 hereafter designated mop1-3. If the ems98941 mutation defines an allele of mop1, then 50% of the complementation cross progeny made with mop1-1 (9-10 individuals) should have a Pl-Rh-like anther phenotype. The observed frequency is not significantly different from this hypothesis (Pearson's goodness of fit x2=0.67; P=0.41) suggesting ems98941 defines an allele of mop1 hereafter designated mop1-4. These assignments are consistent with the complementation results of mop1-3 heterozygotes crossed by mop1-4 homozygotes (Table S3). If the two mutations represent alleles of mop1, then ½ of all progeny (13 individuals) from the two complementation crosses should have a Pl-Rh-like anther phenotype. The observed frequency of Pl-Rh-like types is not significantly different from the expected frequency for this hypothesis (Pearson's goodness of fit x2=2; P=0.16).

Plants homozygous for the ems98287 mutation, having a Pl-Rh-like phenotype, were outcrossed to Pl-Rh/Pl-Rh plants. Almost ½ of F1 plants (8 of 25 individuals from two independent outcrosses) had a Pl-Rh anther phenotype (2 ACS 1; 12 ACS 2; 2 ACS 4; 1 ACS 5; 8 ACS 7) suggesting ems98287 might represent a dominant mutation. However, the Pl-Rh-like phenotype was recovered in 12/40 (30%) F2 plants derived from two independent self-pollinations of ACS 3 F1 plants (3 ACS 1; 19 ACS 2; 5 ACS 3; 1 ACS 4; 12 ACS 7). The observed frequency of F2 mutant phenotypes (30%) is not significantly different from the 25% expected from a single locus recessive mutation (Pearson's goodness of fit x2=0.25; P=0.62) and is consistent with the hypothesis that the dark-anther phenotype, is due to a single locus recessive mutation. Complementation test results (Table 3) indicate the ems98287 mutation complements mutations at rmr2, rmr6, and mop1. If the ems98287 mutation was an allele of rmr1, then ½ of all progeny (24-25 individuals) from the two complementation crosses should have a Pl-Rh-like anther phenotype. The observed frequency of Pl-Rh-like types (20/49) is not significantly different from the expected frequency for this hypothesis (Pearson's goodness of fit x2=0.83; P=0.36), suggesting the ems98287 and rmr1-1 mutations define the same locus. Subsequent molecular mapping of the ems98287 mutation also placed it in the same position as rmr1-1 (bin6.05) thus allowing us to conclude that ems98287 defines an allele of rmr1 hereafter designated rmr1-3.

Using color-converted A619 and A632 inbred lines [2], EMS pollen mutagenesis [1] was repeated and M2 families were screened for the appearance of plants with darkly colored anthers. In separate growouts, the M2 progeny designated 34833 segregated 5/27 plants with ACS 7 anthers. This frequency is not statistically different from the 25% expectation for a single locus recessive mutation (Pearson's goodness of fit x2=0.33; P=0.56) plants with ACS 7 anthers. Pollen from a single ACS 7 plant was used for genetic complementation crosses. This mutation (ems051069) fully complemented mutations defining mop1, rmr2 and rmr6 but failed to complement both rmr1-1 and rmr1-3 (Table S3). If the ems051069 mutation represents an allele of rmr1, then ½ of all progeny (26-27 individuals) from the two rmr1 complementation crosses should have a Pl-Rh-like anther phenotype. The observed frequency of Pl-Rh-like types (28/53) is not significantly different from the expected frequency for this hypothesis (Pearson's goodness of fit x2=0.08; P=0.77), suggesting the ems051069 mutation defines the same locus. The allele defined by the ems051069 mutation is hereafter referred to as rmr1-4.

Pl′ Establishment Tests

Table 1 details crosses done to generate families for genetic tests used to determine the effect of rmr1 mutations on establishment of Pl′ states. Families 05-541 and 05-542 were segregating Pl′ rmr1-1/T6-9 Pl-Rh rmr1-2 plants (FIG. 4). These plants were selected using the dark anther mutant phenotype and then crossed to or by a Pl-Rh homozygous tester. Plants in families 05-103 and 05-539 are the color-converted A632 and A619 testers respectively. The structural genotype (presence or absence of the T6-9 interchange pair) was scored in the field by assaying pollen fertility. The state of the Pl1-Rhoades allele was assayed by evaluating anther pigmentation on a 1 to 7 scale as previously described. Plants with an ACS of 1-4 (representing the Pl′/Pl′ phenotype) are referred to as having light anthers, plants with an ACS of 7 (representing the Pl-Rh/Pl-Rh phenotype) are referred to as having dark anthers, and plants with an ACS of 5-6 phenotype are described as intermediate (FIG. 5).

B′ Establishment Tests

To test the role of rmr1 in b1 paramutation, isogenic B-I and B′ stocks (B1-I; Pl′-Rhoades) were first pollinated by rmr1-1/rmr1-1; b1-W23/b1-W23; Pl1-Rhoades/Pl1-Rhoades plants. Intercrossing the resulting F1 plants (B-I/b1-W23×B′/b1-W23) gave rise to progenies in which there was segregation of Pl-Rh-like and Pl′-like plants. Among these Pl-Rh-like plants, the following b1 genotypes occurred at a 1:1:1:1 frequency: b1-W23/b1-W23; b1-W23/B′; b1-W23/B-I; B-I/B′. Blind test crosses of parental plants displaying dark plant colors (rmr1-1/rmr1-1) were made to b0-CO159/b1-CO159 pistillate testers (Pl-Rh/Pl-Rh; C0159/W23 stock). Actual b1 genotypes of the staminate parents were assigned ex post facto based on segregation of plant color types among resulting test cross progenies (Table 2). An identical strategy using rmr6 mutations has been described.

In Vitro Transcription Reactions

This was carried out as described with husk nuclei isolated from single ears of the same genetic stocks used to measure pl1 RNA differences in anthers (FIG. 7). The anthocyanin genotype of these plants is B1-I (B-I)/B1-I (B-I); Pl1-Rhoades (Pl′) Rmr1/Pl1-Rhoades (Pl′) rmr1-1 and B1-I (B-I)/B1-I (B-I); Pl1-Rhoades (Pl′) rmr1-1/Pl1-Rhoades (Pl′) rmr1-1. The same procedure was applied to single ears of B1-I (B-I)/B1-I (B-I); Pl1-Rhoades (Pl′) Rmr1/Pl1-Rhoades (Pl′) rmr1-3 and B1-I (B-I)/B1-I (B-I); Pl1-Rhoades (Pl′) rmr1-3/Pl1-Rhoades (Pl′) rmr1-3 plants with the following results: four biological replicates pl1, 1.4+/−0.4; b1, 1.3+/−0.6; a1, 6.2+/−1.2 (mean difference+/−s.e.m.).

Alignment of the Helicase Domain of RMR1 with Other Known and Predicted Snf2 Protein Family Members

MUSCLE was used to generate the alignment of the Snf2 helicase domains. The alignment figure (FIG. 8) was shaded with GeneDoc 2.6.03 using a combination of manual and identity modes, with RMR1 being selected as the identity comparison sequence. The lightest level of shading represents conserved residues in at least 60% of the sequences, the middle level is conserved residues is at least 80% of the sequences, and the darkest level is amino acid identity in all sequences.

RT-PCR Expression Analysis

Fully expanded adult leaf (fourth node), flag leaf (terminal leaf), immature tassel, and immature ear tissue was harvested from an adult B73 elite inbred plant 83 days post-germination (FIG. 11). Seedling leaf tissue was harvested from a non-mutant seedling from the F2 rmr1-1 mapping population 10 days post-germination. Seedling root tissue was harvested from a non-mutant seedling from the F2 rmr1-3 mapping population 10 days post-germination. Shoot apical meristem tissue was collected from a seedling (color converted A619 stock) 10 days post-germination. RNA was isolated from the above tissues using TRIzol reagent (Invitrogen, Carlsbad, Calif.) and following the manufacturer's protocol. 10 μg of total RNA was treated with DNase I (Invitrogen) and then reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and Oligo dT(17) primers (Promega). The resulting cDNA was PCR amplified using rmr1 specific primers Os11F and Os15R (Table 4). RT-PCR products sized on a 1% agarose gel and stained with ethidium bromide for visualization. Amplifications of rmr1 from cDNA yields a 640 bp product, while amplification of rmr1 from genomic DNA generates a 2663 bp product (not shown). RT-PCR was also carried on the cDNA using primers specific to alanine aminotransferase (aat) to ensure equal starting amounts of RNA. Amplification of aat cDNA generates a 281 bp product and amplification from genomic DNA produces a 454 bp product. The DNA control was isolated from a color-converted A632 plant as described previously.

Genetic Mapping of Rmr1

The CAPS marker for rmr1-1 was identified by PCR amplifying genomic DNA from the mapping population with primers 1CAPS6F and 1CAPS6R (Table 4) and digesting the product with PvuII (NEB, Ipswich, Mass.). Digested samples represent individuals with the non-mutant polymorphism. For the rmr1-3 population, (134 individuals, 268 chromosomes scored) the primers were 1CAPS10F and 1CAPS10R, the restriction enzyme was BclI (NEB, Ipswich, Mass.), and digested samples represent mutant individuals. No recombinants were found with either marker.

Candidate Gene Selection and Sequencing

See Table 4 for all oligonucleotide primer sequences used. A BLAST search using the rice Os05g326100RF as a query identified maize GSS sequences CG886593, BZ668661, BZ681915, CZ392826, BH878936, CG882444 and sorghum EST sequences AW287235, BG322766, AW285838, BG323020. Sequence was generated for rmr1 from PCR amplicons from various primer pairs. The Os10F/Os10R primer pair amplified sequence identifying the rmr1-3 lesion, and the exonF/exonR amplicons contain the rmr1-1, rmr1-2 and rmr1-4 lesions. To verify the intron/exon structure of cDNA amplified across intron 1 with primers Intron0 F2/Intron0 R2, and across introns 2 and 3 with primers Os11F/Os15R. Additional sequence information was provided by the following primers: mid5′F, Os12F, Os13Rb, intronF, and intronR.

Small RNA Northern Blots

The blots shown in FIG. 8 were carried out as described in the Materials and Methods of the main text with the exception that the DNA oligonucleotides used did not contain doppia sequence, meaning the blots had to be striped and rehybridized with the complementary oligos end-labeled with polynucleotide kinase (Fermentas, EK0031) for accurate sizing.

Example 6 Discussion

RMR1 is the first protein identified whose function acts to maintain trans-generationally repressed states associated with paramutation, a genetic behavior that affects meiotically heritable epigenetic variation through allelic interactions at endogenous loci. The identification of RMR1 as a Snf2 protein highlights an emerging role of these proteins in establishing and maintaining epigenetic marks. In Arabidopsis the Snf2 proteins DRD1 and DDM1 [38,39] are known to maintain cytosine methylation patterns. Lsh1, the mammalian protein most closely related to DDM1, is also required for normal DNA methylation patterns [40-42]. There are some 42 Snf2 proteins in Arabidopsis and at least as many in maize (http://chromdb.org/). This diversity likely represents great functional specialization amongst these proteins. We have placed RMR1 in an RdDM pathway based on its helicase domain similarity to DRD1 and the recent identification of MOP1 as an RDR2 ortholog [14,15]. Consistent with this proposed pathway, the rmr1 mRNA expression profile (FIG. 15) closely matches that of mop1 [15]. Additionally, both RMR1 and MOP1 are necessary to maintain cytosine methylation patterns at silenced transgenes [43], the Pl1-Rhoades doppia sequences, and certain Mutator transposable elements ([15,44]; J. B. H. and D. Lisch, unpublished data). DRD1 is also known to target repetitive elements found in euchromatic contexts through an RdDM pathway [31]. However, the role RMR1 plays to maintain the repressed paramutant states at Pl1-Rhoades appears different than the function of DRD1 in the Arabidopsis RdDM pathway, as RMR1 has, in addition to its requirement for CNN methylation at doppia, a role in the normal accumulation of small RNAs with similarity to that element.

It is unclear how RMR1 mediates the post-transcriptional regulation of pl1 transcripts as suggested by the in vitro transcription and RNase protection assays reported here. It is possible that pl1 transcripts resulting from Pl1-Rhoades in the Pl′ state are less stable than those produced from the Pl-Rh state because of differences in the chromatin environment of Pl1-Rhoades. However, there do not appear to be any Pl′-specific small RNAs produced from the pl1 coding region [12]. In S. pombe it has been shown that the chromatin environment of a locus can affect RNA transcript levels without altering RNA polymerase II occupancy of that locus, leading to the proposal of a cotranscriptional gene silencing mechanism whereby nascent transcripts initiating in a heterochromatic environment are degraded by complexes targeted via heterochromatic small RNAs [17,18]. Chromatin differences in the upstream region of Pl1-Rhoades may favor recruitment of alternative RNA-processing factors or RNA polymerases, which in turn influence the stability of pl1 transcripts. In plants, localization of the large subunit 1a of RNA polymerase IV to loci targeted for RdDM appears necessary for the biogenesis of siRNAs from these loci [28]. When Pl′ repression is disrupted in rmr1 mutants, this alternate genesis or processing of the pl1 transcript may also be lost. Alternatively, our results may highlight a novel role for RMR1-like Snf2 proteins in directly interacting with nascent RNA transcripts via a helicase domain, or in recruiting factors that directly destabilize these transcripts.

Importantly, our analysis of rmr1 mutants calls into question the relationship between RMR1 function and the mechanism of paramutation at Pl1-Rhoades. The mutational screens identifying rmr1, rmr6, and mop1 were designed to discover genetic components necessary to maintain the repressed state of Pl′, not necessarily factors needed to establish this repressed state [10,13]. Therefore, it is possible that loci thus far identified may be indirectly related to the paramutation mechanism. Our results are consistent with a model wherein RMR1 functions in an RdDM pathway, along with an RDR2-like enzyme, MOP1, to maintain a persistent heterochromatic-like chromatin structure at the repetitive element found directly upstream of the pl1 coding region. While it is not clear where RMR1 acts in this pathway it presumably acts coordinately with the maize orthologs of known RdDM components identified in Arabidopsis, namely DCL3 [16,45], the DRM methyltransferases [36], AGO4 [46,47], the RNA polymerase IV subunits, and the maize DRD1 ortholog (FIG. 6A). In this model, doppia transcripts, perhaps because of the repetitive nature of the doppia genomic elements and/or the numerous internal subterminal repeats that are present in these elements [32,48], are the source of aberrant RNA that is processed via MOP1 and a DCL3 enzyme into siRNAs. This small RNA production is carried out in a manner that is dependent on RMR1 activity, possibly via direct interaction with a small RNA processing complex or by making the DNA accessible to factors necessary for siRNA precursor generation such as polymerase IVa. These siRNAs, through the activity of AGO4, DRM enzymes, and polymerase IVb, then establish a heterochromatic state at the Pl1-Rhoades doppia-like element that is present in both Pl-Rh and Pl′ states. The methylation effects seen in rmr1 mutants might indicate that this heterochromatization machinery depends on the activity of RMR1 to feed back on the doppia element, or loss of RMR1 may short circuit this pathway and thus affect methylation activity indirectly. An RMR1 defect then affects stability of paramutant states at pl1 because of the chromatin context of the Pl1-Rhoades allele, and not through direct disruption of components required for paramutations to occur. This is in line with a report that MOP1-dependent small RNAs produced at the b1 locus are insufficient to mediate paramutation [49].

The relationship between RMR1 action, the chromatin organization of Pl1-Rhoades, and the repressed Pl′ state is not clearly understood at this time. It is possible that derepression of the upstream repetitive element makes the region more accessible to general transcription factors whose actions could destabilize repressive Pl′ chromatin states that are independent of those maintained at doppia (FIG. 6A). Indeed, RNA polymerase processivity can lead to changes in the chromatin environment through histone modifications or histone replacement [50,51]. Alternatively, Pl′ chromatin states may represent a spreading of the heterochromatic domain at doppia into a euchromatic region defined by the Pl1-Rhoades gene space (FIG. 6B). In fission yeast, heterochromatic domains nucleated by small RNAs have the ability to spread in cis through successive H3 K9 methylation [52]. In this situation, loss of RMR1 function would alleviate Pl′ repression by disrupting maintenance of this expanded heterochromatic domain. In either of these situations RMR1 affects Pl1-Rhoades paramutations by virtue of its role in maintaining heterochromatic states at a proximal repetitive element.

McClintock was the first to describe derivative alleles in which transposons acted to control the expression patterns of attendant genes [53]. It is now clear that epigenetic modulations of the transposons themselves—what McClintock referred to as “changes in state”—can alter the regulatory properties of individual genes both somatically [54] and trans-generationally [55,56]. Our results indicate that even transient changes in state of the Pl1-Rhoades doppia fragment can have trans-generational effects on pl1 gene expression patterns. These experimental examples, in the context of McClintock's thesis [53], point to a dynamic source of regulatory, and potentially adaptive, variation adjunct to the DNA itself. Precisely how this epi-variation relates to existing genome structure and function, as well as its evolutionary potential, remains a largely unexplored area of investigation.

Currently, well-characterized examples of paramutation are limited to loci where expression states have a clear phenotypic read-out, such as pigment synthesis. cis-Elements required to facilitate paramutation have been functionally identified at specific alleles of b1 and colored1 (r1) [57-59]. To date, there is no evidence that the chromatin status of these cis-elements is affected by mutations at trans-acting loci required for maintenance of repressed paramutant states. It appears that paramutations represent a type of emergent system wherein genomic context and maintenance of chromatin states interact to facilitate meiotically heritable epigenetic variation. In this view, it is possible that cis- and trans-elements necessary for maintenance of such variation might not interact in a direct and predictable manner. What remains to be seen is the extent to which this type of system acts throughout the genome. Genome-wide screens for paramutation-like behavior, in which expression states are affected by allele history, remain technologically and conceptually challenging. Recent work by Kasschau et al. [60] suggests that in Arabidopsis, few endogenous genes are regulated by proximal presumed RdDM targets. However, it is tempting to speculate that examples of paramutation represent an exception to this trend, representing a mechanism by which populations can quickly, and heritably, change their transcriptome profile and regulation.

Example 7 RMR1 Function Contributes to Inbreeding Depression

Our genetic and molecular studies show that Pl1-Rh favors a reduced activity state when maintained in homozygous condition [9] and that a RdDM/CTGS pathway involving RMR1 maintains this repressed state [65]. If significant numbers of alleles show behaviors similar to Pl1-Rh, then inbreeding could lead to widespread genetic repression affecting plant growth, development and homeostasis. The fact that rmr1 mutants have vastly reduced levels of genome-derived ˜24 nt RNAs (FIG. 16) suggests many alleles that would otherwise be repressed by the RMR 1-dependent pathway would remain active. Hence inbreeding syndromes might be mitigated to some extent in rmr1 mutant lineages. For initial genetic mapping, the rmr1-1 mutation was crossed to the A632 inbred and recovered in homozygous condition from an F2 family. Three lines of single seed descent were initiated in the S1 progeny and it was surprising to find that plant quality remained fairly consistent in S2-S11 generations; silking and anthesis remained coincident, pollen shed remained copious with no evidence of aborted grains above the typical ˜5% level, plant morphology appeared unaffected aside from mild stunting, and seed set was consistently full. This was unexpected as, in our experience, typically few recombinant inbred lines remain suitable for propagation past the S4 generation and most other rmr-class mutants display severe degradations in plant quality upon selfing [13, 66; J Hollick, unpublished]. As a control, parallel lines were developed from S1 progeny of a single Rmr1-A632/rmr1-1 individual from the same initial F2 mapping progeny in order to compare inbred Rmr1-A632/Rmr1-A632 versus rmr1-1/rmr1-1 genotypes. At the S5 generation, lines of both genotypes were planted in adjacent plots and visually evaluated for general plant characters and subsequent seed set on selfed ears. Plants from the four established rmr1-1/rmr1-1 lines were uniform and unremarkable in type, and selfed ears from all 39 plants had near full seed set. In contrast, 9 of 24 plants from the two Rmr1-A632/Rmr1-A632 lines (3/9 and 6/13 off-types in the respective lines) were phenotypically abnormal. Four of these 9 off-types were classified as “runts” as their heights were less than ˜⅓ of their siblings and one of these had conspicuously narrow leaf blades. None of these four runts produced silking ears. Four plants had delayed silking relative to pollen shed; two of these plants produced tiny ears with no grains and two plants had normal sized ears with only a single grain each. One otherwise normal plant had vestigle apical leaves and no apical inflorescence. Thus only ˜55% of the inbred plants with normal RMR1 function were phenotypically normal but all the inbred plants derived from the four founder rmr1-1/rmr1-1 mutants were of good quality similar to that seen in the original set of S2-S11 rmr1-1/rmr1-1 lines. These observations combined suggest that loss of RMR1 function helps prevent the degradation of plant quality typically seen in inbreeding depression.

Example 8 The Rmr1-1 Mutation Affects Transgressive Traits Manifest in Heterotic Crosses

As heterosis and inbreeding depression are often thought to be opposite manifestations of a fundamentally similar genetic relationship, it seemed reasonable to test whether or not preconditioned epigenomes (+/−RMR1 function) would respond differentially in heterotic crosses. Two ears from the same A632, Mol7, or B73 plants were differentially pollinated using single pollen sources from S4 Rmr1-A632/Rmr1-A632, and S4 rmr1-1/rmr1-1 plants and small progenies (16-20 individuals) were evaluated for several traits including dry ear weight. Results of this initial test were striking (Table 5) as at least one non-anthocyanin trait was differentially affected in every cross. Most significant was the observation that dry ear weight was nearly 10% greater when the single B73 parent received pollen from the rmr1-1 line versus the Rmr1-A632 line. There are at least two major experimental concerns with this exciting result. First, this simple measurement does not control for differences in grain moisture and includes the cob, typically discarded in such yield evaluations. Secondly, and most problematic, the contrasting lines are only ˜75% genetically identical. Ideally, these tests would use nearly isogenic parents (see Example 12).

These two sets of preliminary data regarding the differential breeding behaviors of rmr1-1/rmr1-1 and Rmr1-A632/Rmr1-A632 plants are consistent with a hypothesis in which genomes conditioned in the presence or absence of RMR1 function have unique epigenomic landscapes that dictate differential genic action or dynamic responses. Published data regarding Pl1-Rh action validates this hypothesis at a single locus level.

Example 9 Evaluate Rmr1 Knockdown (KD) Approaches to Mitigate Transgene Silencing

RNAi-based strategies will be tested on existing silenced transgenes and newly developed GFP/GUS reporter constructs in transgenic maize plants. Given the observations that rmr1-1 mutations are able to release transcriptional repression of a silenced 35S-B 1 transgene array [43], it is quite likely that RNAi-based knockdown (KD) lines of rmr1 RNA could be broadly employed to mitigate, or control, transgene silencing for commercial application. The fact that RMRI defects themselves appear not to affect plant development [10] suggests that such KD strategies can complement existing breeding programs without compromising biomass yields. As an experimental overview, CAMBIA-based vectors will be used to create both RMR1 KD and promoter-GFP reporter constructs, and fertile transgenic maize lines will be established to compare effects of RMR1 on existing and de novo reporter gene silencing.

Create and Validate Rmr1 Knockdown Lines

We will generate public lines that can be used to release and control transgene silencing. The NSF-sponsored maize chromatin consortium (NSF DBI-0421679; www.chromdb.org) has had good success using bioinformatics and subsequent double-stranded RNA (dsRNA) based constructs to molecularly knock-down RNA expression of specific endogenous maize genes even when they belong to multigene families ([43]; 26 individual genes validated for molecular KD phenotypes). Unfortunately, rmr1 was not among the ChromDB KD targets since it was, to this point, not represented in available EST collections. The pMGC161 vector and cloning technology developed in the maize chromatin consortium will be used to create a dsRNA expression construct specific for rmr1 (double CaMV35S promoter driving an inverted repeat of −1 kb of Rmr1 exon3 separated by a O. sativa waxy1 intron stuffer). Recombinant DNA manipulations will be carried out in E. coli and the sequence-validated construct will be electroporated into A. tumefacians strain EHA101. With appropriate APHIS permit, a transformed Agro strain will be shipped to the Iowa State Plant Transformation Facility (PTF) for maize (Hill line) transgenesis. We will request that PTF provide seeds from 8 separate glufosinate-resistant transformation events. Transgene-based effects on endogenous Rmr1 RNA levels will be assayed using RT-PCR, and Southern blotting will be employed to characterize insert structures for those plants demonstrating a molecular KD phenotype. Agro-mediated transformation will be chosen to establish these lines as there is a greater chance for single inserts of low transgene complexity that, in general, tend to escape gene silencing. If Rmr1 knockdowns help mitigate this type of general repeat-induced silencing, then transgenesis methods and insert complexity may be inconsequential. Molecularly-vetted lines will be advanced to specific transgene silencing tests as described in the following section and will be crossed to a B73 T Pl′ line [a T6-9 (043-1) interchange with the Pl1-Rh allele of Pl′ state −1.5cM from the breakpoint [11] introgressed (A) to −94% B73] to test effects on maintaining Pl′ paramutant states. We fully expect that any functional KD lines will phenocopy the existing twirl mutations with this assay. Using the same B73 T Pl′ line as recurrent female, 2 functional Rmr1 RNAi transgene inserts will be backcrossed for subsequent deposit to the Maize Genetics Cooperation Stock Center (Urbana, Ill.). As the Hill line is a B73 5 A188 hybrid, introgression to >90% B73 will be swift. The T Pl′ interchange may prove useful for specific line developments by other researchers [11] but can just as easily be purged.

Test Effects of Rmr1 RNAi on a Silenced pWRWR Transgene Array

Prior results indicate that the rmr1-1 mutation mitigates transgene silencing both somatically [43] and sometimes meiotically (FIG. 17). As such, we expect that production of dsRNA specific to Rmr1 will similarly lead to the reactivation of the silenced pWRWR transgene array [67]. Although the test is genetically simple, both Rmr1 RNAi and pWRWR transgene loci are linked to a glufosinate-resistance marker. Fortunately, we have introgressed the silenced pWRWR transgene into a 4Co63 T Pl′ line (null for pericarp color1 function) so that a dark anther phenotype is a proxy for Rmr1 knockdown (FIG. 18) and pericarp coloring of any kind reflects pWRWR transgene expression (FIG. 17). With these visual features, we can evaluate large numbers of individuals with each transgene combination to determine whether or not the RNAi constructs are effective at releasing silencing of the pWRWR repeated array. A molecularly vetted Rmr1 KD plant will be crossed with a plant hemizygous for the silenced pWRWR insert and −300 progeny grown to maturity. Individuals will be visually scored for anther phenotypes and each plant will be pollinated with the 4Co63 T Pl′ line so that follow-up evaluations of meiotically-heritable reactivations can be documented in the next generation. Hand-harvested ears will be tagged and visually inspected for pericarp color. For those ears showing evidence of pWRWR reactivation, progeny sets will be grown out and similarly assayed. Additional backcrosses can be used to follow the stability of reactivated states for several more generations.

Test Effects of rmr1 RNAi on de novo Transgene Silencing

While Rmr1 KD lines promise to be extremely useful for controlling transgene function within the broader research community, the largest economic potential would appear to reside in mitigating de novo transgene silencing during the transgenesis procedure itself. Commercial lines are now so far removed from the academic inbreds used by most maize geneticists that an introgression-based plant improvement strategy is unattractive. Using Hill as a representative, we will compare PTF-generated transgenesis events with cell autonomous GFP/GUS-reporter constructs in the presence or absence of dsRmr1-generating constructs using biolistic co-bombardments of embryogenic callus and subsequent plant regeneration. We will evaluate locus and copy numbers using Southern blot analyses and measure relative reporter GUS expression levels in TI plants and reevaluate these features following outcrossing to our B73 T Pl′ stock. We expect, as before, that any functional Rmr1 KD transgenes will result in visual reactivation of Pl′ states (darkly colored anthers). Analysis of the outcross progeny will be important to verify locus numbers, transgene linkages, and to assess both the genetic and epigenetic stability of the reporter gene arrays.

The Rmr1 dsRNA construct has been described in above. The reporter constructs will be derivatives of the pKGWFS7 binary vector [68] in which a recombinase mediated insertion of promoter sequences (Gateway technology; [69]) drives the expression of an endoplasmic-retained GFP-GUS fusion protein. To leverage existing resources and provide reagents for future studies, we will make separate constructs using the double CaMV35S promoter, −2.5 kb of pl1-B73 [70], and as much unique 5′ sequences we can obtain from rmr1-B73. Based on unpublished sequence comparisons (S. Gross and J. Hollick), the immediate −2.5 kb region of pl1-B73 is unique from the nearly 8 kb of 5′ sequences we have of pl1-Rhoades, so do those sequences confer the same tissue-specific patterns of pl1-B73 expression as displayed by pigment patterns, or are some of the sequences shared with pl1-Rhoades important to tissue-specific regulation? The rmr1-B73 reporter fusion will help us understand Rmr1 gene control and will complement subsequent proteomic studies by highlighting tissues of potentially high protein expression and of functionally important significance. More details regarding the Rmr1-B735′ sequences can be found in Example 8. All promoter regions will be PCR-amplified, introduced into a Gateway topoisomerase-based cloning vector [71], sequenced, and shuttled into pKGWFS7 for biolistic transformation. Reporter constructs will be co-bombarded with either the Rmr1 RNAi construct or empty pMGC161 vector. We will request TI seeds from 6 events from each biolistic combination for a total of 36 lines.

Example 10 Identify RMR1 Molecular Partners Using Affinity Purification, Mass Spectrometry, and cDNA Sequencing

Macromolecular complexes-affinity purified from transgenic plant expressing functional TAP-tagged RMR proteins—will be fractionated and analyzed using mass spectrometry and cDNA sequencing. While our forward genetic screens have been successful at identifying molecular components required to maintain Pl′ states, there are undoubtedly other components that can only be identified using biochemical or proteomic approaches. One of the major advantages of studying the mechanics of gene silencing in a physically large organism like maize is the abundant availability of defined tissues for use in biochemical purifications. In theory, one could identify RMR1 interacting proteins using a yeast 2-hybrid (Y2H) assay with a maize cDNA library. To our knowledge, there is currently no cDNA library available in which an Rmr1 sequence has been identified. Further, the biological relevance of interactions detected in the Y2H are often unknown, or questionable when compared to affinity purification of in vivo complexes followed by proteomic analysis [72]. The Y2H assay thus appears better suited to verification studies than as a primary screening method. There is no simple approach; therefore one should maximize the likelihood that any detected interactions are biologically relevant. Tandem-Affinity Purification followed by Mass Spectrometry (TAP-MS) has recently emerged as a powerful methodology for identifying protein-protein interactions in monocots [73]. By complementing a rmr1-3/rmr1-3 defect with a TAP-tagged RMR1 protein expressed from the Rmr1-B73 promoter, we will purify functionally relevant macromolecular complexes for RNA and proteomic analysis. With this approach we will discover novel components of the maize gene silencing machinery and, in turn, provide new reagents, targets, and models for future studies.

Create Monocot Expression Constructs for RMR1-TAP Fusion Molecules

Since expression from a constitutive promoter like ubiquitin might lead to non-biologically relevant protein associations in cell or tissue types in which Rmr1 is not typically expressed, we would like to have the native promoter drive a RMR1-TAP fusion protein. We will therefore modify a pCAMBIA1300-based Gateway vector [74] to have a 5′ portion of the Rmr1-B73 sequence replace the resident ubiquitin promoter. This sub aim requires some additional vetting of the Rmr1-B73 gene structure to identify the transcription start site and non-repetitive 5′ putative promoter sequences. We will also need to generate a near full length Rmr1 cDNA to create the in-frame translational fusions with the TAP tag. We will begin with a N-terminal TAP addition (NTAP) and will modify the construct as needed to create a CTAP version.

According to a multiple species alignment, of almost exclusively bioinformatics-based annotations, the RMR1 protein should begin at the presumed translational start site (FIG. 19). We are currently using 5′ rapid amplification of cDNA ends (5′ RACE) to identify whether any additional protein coding sequence is present in Rmr1 mRNAs. We will also use a combination of primer extension and RNase protection assays to verify and assign the transcription start site. We will subclone and sequence 5′ flanking sequence from the in-house B73 BAC and use BLAST to survey up to ˜4 kb of 5′ sequence for transposons and gene fragments that might help delimit intergenic regulatory sequences. We will use restriction-enzyme based strategies to swap the ubiquitin promoter in the binary vector with as large a piece of the Rmr1-B735′ sequence as possible. Based on the 5′ RACE results, we will design appropriate primer sets to amplify the protein-coding portion of a RT-derived cDNA molecule and introduce these amplicons into Gateway topoisomerase-based cloning vectors [71]. These clones will be sequenced and shuttled into our pCAMBIA1300-Rmr1 promoter derivatives for subsequent A. tumefaciens mediated maize transformation.

RMR1-TAP Functional Complementation

To maximize the likelihood that an affinity purification strategy will identify functionally relevant RMR1 protein partners, it is preferred that the tagged RMR1 protein is functional and not in competition with native RMR1 protein. Therefore, we will test transgenic complementation of the rmr1-3 putative null mutation using the C and N-terminal tagged constructs (described in the section above). Transgenic plants exhibiting rmr1-3 complementation will be used for tandem affinity purification of RMR1-TAP complexes (see section below). With APHIS permits, A. tumefaciens strain EHA10-transformed with the binary vectors detailed in SA2a—will be forwarded to PTF, and we will request seeds from 10 independent transformation events for each construct. Rapidly dividing tissues of glufosinate-resistant Ti plants will be assayed by western blots for expression of the proteinA component of the TAP tag, and positive plants will be crossed by our B73 homozygous T Pl′rmr1-3 line. Glufosinate-resistant, protein A-positive progeny will be crossed by a B73 T Pl′rmr1-31T Pl′Rmr1-B73 heterozygote to ensure that all progeny receive a pl1-Rhoades allele of P1′ state. If the RMR1-TAP constructs genetically complement the rmr1-3 defect, then glufosinate-resistant plants with fully fertile pollen (T/7) and a rmr1-3/rmr1-3 SNP-genotype should have a distincT Pl′ anther phenotype (FIG. 18). These same genotypes will have Pl-like anthers if there is non-complementation. If the large (181 aa) N- or C-terminal TAP addition does not allow proper folding or protein function, this strategy may not work. Even so, we expect these lines will be useful for assessing intracellular localizations via TAP-based immunocytochemistry.

Identify RNA and Protein Partners Using Tap-Based Purification, Biochemical Fractionation, Nucleic Acid Sequencing, Mass Spectrometry and Proteomics

Guided by the expression profiles revealed by the promoter-reporter constructs (described in Example 7), or indicated by proteinA-based western blotting, TAP-based purifications [73], total protein quantifications, and SDS-PAGE will be used to assess purification, yields, and numbers of complex members. We will perform the same purification steps on untransformed plants as a purification-specificity control. Based on these estimates, we can appropriately scale subsequent production purifications. We do not expect plant material to be limiting for these purifications. Depending on the number of complex members, we will repeat purifications and provide desalted trypsinized 500 ng of protein for nanoscale reverse-phase (RP) HPLC (1-10 proteins), or 100 μg for ion exchange and RP HPLC (>10 proteins), separations followed by electrospray ionization with tandem MS/MS detection. The Cancer Research Laboratory at the University of California at Berkeley will provide both the mass spectrometry and Seaquest-based protein identification on a recharge basis. The facility runs a Thermo Finnigan LCQ Decca XP Plus ion trap mass spectrometer with a NanoLC/ESI ion source for identification of peptides in complex mixtures. Data analysis will be performed by an IBM 16 node computational cluster.

Given DRD1 may interface directly with siRNAs or indirectly via complex association with AGO4 [28], it would not be unexpected to find RMR1 complexed with small RNA molecules [75]. If small RNAs co-purify with TAP-tagged RMR1, we will fractionate these on polyacryamide gels, excise and prepare for amplification and small-scale sequencing. Should there be significant RNA sequence heterogeneity within these samples, we will perform deep sequencing of these RNA populations using 454 or Solexa platforms.

Example 11 Measure Trait Variances Associated with RMR1 Function in Heterotic Crosses

Near-isogenic inbred lines will be tested for heritable effects of RMR1 function on traits of commercial importance. While well documented that rmr1 mutations lead to heritable changes of Pl1-Rhoades function and hence pigment traits [10], effects on alleles affecting other traits remains speculative. Given preliminary data showing inbred genomes conditioned in the absence of RMR1 function increase progeny dry ear weights nearly 10% in crosses to the B73 inbred (Table 5), a more rigorous test of the epigenetic complementation concept will be conducted. As an overview of the proposed experimental design, relative grain yield, days-to-flowering, and plant height will be compared among progeny of A619 5 A632 and B73 5 Mol7 reciprocal crosses in which one or both parents is deficient for RMR1 function. Both rmr1-1 and rmr1-3 alleles, introgressed (A) to ˜97% into the above-mentioned inbred lines, will be tested initially.

Germplasm Development

The A619, A632, B73, and Mol7 elite inbred lines were originally obtained directly from the North Central Plant Introduction Station (Ames, Iowa). Prior to molecular identification, rmr1 mutant alleles had been committed to an inbred backcross program requiring concomitant introgression of a reporter Pl1-Rhoades allele linked to the T6-9 (043-1) breakpoint. Molecular SNP-based genotyping will rapidly advance these introgressions in the absence of the translocation chromosome so that the A619/A632 materials can be combined. A comparison of the helicase-domain (rmrl-1), and predicted null (rmrl-3) mutations will be important to assess possible loss-of-function strategies for commercial application. These efforts will provide highly introgressed rmr1 mutant lines that will be deposited to the Maize Genetics Cooperative Stock Center for curation and dissemination to the broader research community.

Heterotic Crosses

Respective combinations of inbred lines B73 or A632 (representative of Reid Yellow Dent) and Mol7 or A619 (representative of Lancaster Sure Crop) represent one of the most highly utilized heterotic patterns in the commercial hybrid industry. As Pl′ reversion tests suggest a parent of origin effect [10], it is desirable to sample rmr1-conditioned genomes through both pistil and pollen transmission. For each BC4F2 progeny, plants homozygous for either inbred Rmr1 or rmr1 mutant alleles will be identified by SNP-typing and reciprocally crossed with plants from the inbred heterotic parent (i.e. rmr1-1 A619 5 A632; A632 5 rmr1-1 ^ A619; A619 5 rmr1-1 ^ A632; rmr1-1 ^ A632 5 A619).

Sampling and Measurement

Five ear-to-row plots will be evaluated from each heterotic cross for days-to-flowering, plant height at anthesis, and dry kernel weights. For each plot, three 20-kernel rows will be planted side by side and, in an attempt to simulate large field conditions, only plants of the interior rows will be sampled. Each of the 5 genotypic complements (complete set of crossing combinations) of test plots will be assigned field placements in a randomized design to effectively neutralize environmental variations and will all be planted on the same day. Plots will be open pollinated and individual ears will be hand-harvested, dried in a 100° F. room briefly to minimize kernel damage and shelled for weight measurements. Kernels will be further dried to 12-15% moisture content and the kernels per plant will be directly weighed on a Federal Grain Inspection Service-approved balance. Test weights (lbs/bushel) per plot will be approximated using a hand-held balance. Grain yield estimates of Mg per hectare will be calculated per plot and per genotype. Mean values+/−s.e.m. for each set of 5 test plots will be compared using a two-tailed z-test. Since it is quite likely that not all sampled plots will contain 20 plants, an appropriate scaling factor will be applied to obtain mean yield estimates.

Should these initial studies with A619 and A632 reveal biomass and/or grain yield increases, this experimental design will be repeated with rmr1 mutations introgressed to ˜99%, and we will forge collaborations for field-based yield trials. If no differences are indicated, we will still replicate the experimental design with B73 and Mol7 combinations. As it is possible that epigenome conditioning is progressive and cumulative, the experimental design will be repeated using appropriate BC4F2S3 A619 and A632 lines. Given the pedigree of these materials, it is expected that provenance tests, measurements of these or additional traits, and statistical treatments can be used to make estimates of epigenetic variation contributions to broad-sense heritability. Recombinant inbred lines will also be derived from these materials to measure the effects of RMR1 defects on inbreeding depression in future projects.

Example 12 Monitor In Vivo RMR1-Based Silencing Behaviors

A RMR1 RNAi-based knock-down construct will be evaluated for mitigation of transgene silencing and the cell autonomy and resilencing properties of RMR1 action will be measured at the boundaries of irradiation-induced segmental monoploid sectors. Given that the rmr1-1 mutation is able to release repression of silenced 35S-B1 [43], pWRWR, and 35S-H2B-YFP transgene arrays, it is quite likely that RNAi-based knockdown (KD) lines of Rmr1 RNA could be broadly employed to mitigate, or control, transgene silencing for commercial application. The fact that RMR1 defects themselves appear not to affect plant development [10] suggests that such KD strategies can complement existing breeding programs without compromising yields. The experiments will generate data regarding efficacy, cell autonomy, and kinetics of RMR action on transgene silencing. These efforts will generate materials immediately translatable to crop improvement efforts.

Create and Validate Rmr1 Knockdown Lines

The pMGC161 vector and cloning technology developed in the NSF-sponsored maize chromatin consortium ([43]; NSF DBI-0421679; www.chromdb.org) will be used to create a dsRNA expression construct specific for Rmr1 (double CaMV35S promoter driving an inverted repeat of ˜1 kb of Rmr1 exon2 separated by a O. sativa waxy1 intron stuffer). Recombinant DNA manipulations will be carried out in E. coli and the sequence-validated construct will be electroporated into A. tumefacians strain EHA101. With appropriate APHIS permit, the transformed strain will be shipped to the Iowa State Plant Transformation Facility (PTF) for maize (HiII line) transgenesis. We will request PTF provide seeds from 10 separate glufosinate-resistant transformation events. Transgene-based effects on endogenous Rmr1 RNA levels will be assayed using RT-PCR and Southern blotting will be employed to characterize insert structures for those plants demonstrating a molecular KD phenotype. Agro-mediated transformation is chosen to establish these lines as there is a greater chance for single inserts of low transgene complexity that, in general, tend to escape gene silencing. If Rmr1 knockdowns help mitigate this type of general repeat-induced silencing, then transgenesis methods and insert complexity may be inconsequential. Molecularly-vetted lines will be advanced to specific transgene silencing tests (see Example 7) and will be crossed to a B73 T Pl line [a T6-9 (043-1) interchange with the Pl1-Rh allele of Pl state ˜1.5cM from the breakpoint [11] introgressed (^) to ˜97% B73] to test effects on maintaining Pl paramutant states. We fully expect that any functional KD lines will phenocopy the existing rmr1 mutations with this assay. Using the same B73 T Pl line as recurrent female, 2 functional Rmr1 RNAi transgene inserts will be backcrossed for subsequent deposit to the Maize Genetics Cooperation Stock Center (Urbana, Ill.).

Test Effects of Rmr1 RNAi on Silenced Transgene Arrays

Although rmr and mop mutations alone can suffice to mitigate transgene silencing (U.S. Pat. No. 7,264,970), commercial lines are now so far removed from academic inbreds used by most maize geneticists that an introgression-based plant improvement strategy is unattractive. Data regarding de novo mitigation of transgene silencing with RMR1 RNAi KD technology is expected to motivate this approach for agronomic applications. Based on preexisting mutant analyses we expect that production of dsRNA specific to Rmr1 will similarly lead to the reactivation of both silenced pWRWR and 35S-H2B-GFP arrays. The partially silenced 35S-H2B-GFP line has been made available from our collaborator for these studies. Although the tests are genetically simple, both Rmr1 RNAi and silenced transgene loci are linked to a glufosinate-resistance marker. Fortunately, we have introgressed the silenced pWRWR transgene into a 4Co63 T Pl line (null for pericarp color1 function) so that a dark anther phenotype is a proxy for Rmr1 knockdown and pericarp coloring of any kind reflects pWRWR transgene expression. Marker-assisted selection will be used to synthesize 35S-H2B-GFP/RMR1 RNAi KD lines. With the visual phenotypes afforded by the various transgenes, we can evaluate large numbers of individuals with each transgene combination to determine whether or not the RNAi constructs are effective at releasing silencing of the repeated arrays. A molecularly vetted Rmr1 KD plant will be crossed with plants hemizygous for the silenced pWRWR or 35S-H2B-GFP inserts and ˜300 progeny of each will grown to maturity. For the pWRWR lines, progeny individuals will be visually scored for anther phenotypes and each plant will be pollinated with the 4Co63 T Pl line so that follow-up evaluations of meiotically-heritable reactivations can be documented in the next generation. Hand-harvested ears will be tagged and visually inspected for pericarp color. For ears showing pWRWR reactivation, progeny sets will be grown out and similarly assayed. For the 35S-H2B-GFP lines, progeny will be PCR-genotyped and then tissues of selected individuals will be evaluated by epi-fluorescence microscopy using standard dissecting scope equipment available in the Hollick/Amacher lab. Follow up trans-generational studies will be used to measure transmission properties of these reactivated transgenes.

Use Mosaic Analysis to Test Cell Autonomy and Kinetics of Rmr1 Function

Given the structural and molecular genetic similarities of RMR1 to CLSY1, it is possible that some RMR1-mediated silencing may be non-cell autonomous. This possibility can be addressed using genetic mosaics. We have used mosaic analyses to show that Pl changes to a Pl state progressively in Pl/Pl heterozygotes. From these sector boundaries, visual inspection of anthocyanin pigment levels indicate that paramutagenic action is cell autonomous. Similar boundary analyses are proposed to test the cell autonomy, silencing, and reactivation properties of RMR1 on the partially silenced 35S-H2B-YFP transgene array (FIG. 20). The following cross will be used to create progeny genotypes that will be subjected to mosaic analysis; 1) 35S-H2B-YFP; Rmr1/Rmr1 w15 rmr1-1/+rmr1-1. This progeny can be generated immediately using the partially silenced 35S-H2B-YFP line and a w15 Pl rmr1-1/+Pl Rmr1 line we have already synthesized. Using the same irradiation protocol as detailed in Hake and Sinha (1994) and as used previously, in situ YFP-fluorescence will be documented at boundaries marked by segmental monoploid albino tissue (35S-H2B-YFP; w15 rmr1-1/ - - - ). Evaluating reactivated YFP fluorescence coincidence with sector boundaries will indicate whether or not the silencing function of RMR1 is cell autonomous. As dry seed-induced sectors occur during a developmental window, there will be an opportunity to observe these clonal reactivation events in a broad range of sector sizes and developmental timings. In addition to testing the CLSY1-like properties of facilitating intercellular silencing, we expect these studies will provide basic information needed to evaluate future transgenic approaches in which RMR1 functions are manipulated for tissue-specific control of silencing.

Example 13 Characterize RMR1 Biochemical Functions

To functionally classify the type of biochemistry carried out by this novel Snf2 protein, recombinant RMR1 will be characterized with in vitro assays of ATPase, nucleic acid translocation, and nucleosome sliding activities. It is unknown how RMR1 affects the stability of pl1 RNA and small RNA abundance, two phenotypes not observed in any biochemically characterized Snf2 protein. Although RMR1 contains structural motifs for ATP binding and hydrolysis, it is also unknown if RMR1, or related proteins CLSY1 and DRD1, is a functional ATPase. We will purify recombinant RMR1 and derivatives from a heterologous protein expression system and assay these for characteristic Snf2 protein functions: nucleic acid-stimulated ATPase activity, ATP-dependent dsDNA translocation, and nucleosome sliding. These kinetic measurements of RMR1 action are essential to understanding the molecular role of RMR1 in co-transcriptional repression, small RNA biogenesis, and RdDM. These efforts will provide recombinant RMR1 proteins with which to develop additional immunoreagents, protein interaction assays, and crystals for future studies.

Prepare Recombinant Proteins

DNA clones containing SsoRAD54 and the E563Q mutant [22] will be obtained and used to construct controls in all assays. We will express and test both full-length protein (RMR1fl; 161 kDa) and a truncated form (RMR1cd; ˜70 kDa) containing the most highly conserved Snf2 region. Full length and truncated Snf2 proteins have been successfully expressed and purified from both prokaryotic and baculovirus systems [E. coli: 76, 22, 77; baculovirus: 39, Gaillard et al. 2003, 78], and truncated proteins containing only the conserved domains exhibit full function in vitro assays [76,22]. These assays will also be used to characterize the biochemical defects of RMR1-1, RMR1-2, and RMR1-4 derivatives that contain amino acid substitutions predicted to disrupt nucleic acid binding and ATP hydrolysis. Coding sequence for RMR1fl and RMR1cd will be amplified (using maize flcDNA) and ligated into pET302 and pET303 vectors (Invitrogen) containing a T7lac promoter and either N or C-terminal hexahistidine (His) tags respectively. Recombinant expression vectors will be transformed into BL21-CodonPlus RIL competent cells (Stratagene) that contain extra copies of E. coli argU, ileY, and leuW tRNA genes to reduce the negative effects of codon bias on heterologous protein expression. His-tagged proteins will be purified on Ni-resin columns under native conditions and prepared for individual assays.

Perform Biochemical Assays

ATPase Assays:

ATPase assays measuring radioisotope release from −³²P ATP will be performed with double and single stranded DNA substrates [22,79]. Given the role RMR1 plays in RdDM, it will be important to measure dsRNA, ssRNA and RNA:DNA duplex stimulation. Tests of the RMR1 mutants are expected to verify predicted effects of the mutations on ATP hydrolysis and they will serve as a negative control to show specificity of the assay for RMR1. The controls used in this assay either have dsDNA dependent ATPase activity (SsoRAD54cd) or are inactive (DExx box disrupting E563Q; 22). Titrating reaction component concentrations (such as ATP, protein, and nucleic acids) will allow detailed measurement of wild type and mutant RMR1 kinetics to compare with those measured for SsoRAD54cd and other Snf2 proteins representing the major functional classifications [80, 81, 77, 22, 76, 79].

Translocation:

Snf2 proteins are predicted to use ATP hydrolysis to translocate along dsDNA, and this translocation activity is routinely assayed by efficient removal of triplex forming oligonucleotides (TFO). TFOs tightly bind the major groove of DNA double helices to promote alterations in gene expression [82]. TFO removal assays will be performed as described [22,83] using triplex DNA formed by binding of a ³²P-labeled TFO to the corresponding binding site on duplex DNA [83]. Free TFO is detected by gel separation and quantified by phosphorimage capture and image processing.

Nucleosome sliding: In vitro mobilization of mononucleosomes is thought to reflect a chromatin remodeling function in vivo [84]. This assay is preferred over nuclease sensitivity assays because it produces cleaner results by using small, well-defined chromatin templates. Radiolabeled mononucleosomes are generated by serial dilution transfer from chicken oligonucleosomes onto ³²P body-labeled DNA templates and verified by native PAGE [85]. Purified mononucleosomes will be incubated with RMR1, and at specific time points (1-60 min), aliquots are removed and the reaction quenched. Nucleosome boundaries are then assayed by ExoIII mapping. Nucleosome mobility is determined by the presence of a ladder of partially degraded DNA products [39,86]. SsoRad54cd will be included as a positive control, and the RMR1 mutants will also be assayed for nucleosome mobilization ability.

Example 14 Define RMR1 Subcellular Localizations and Epistatic Relationships

To define both the intracellular and epistatic positions of RMR1 within the nucleus and RdDM/CTGS pathway, materials will be synthesized for immunofluorescence detection of functional RMR1 in normal and rmr/mop mutant backgrounds. These materials will also be important for basic RMR1 protein characterizations and for future biochemical purifications and interaction studies of RMR1 protein partners. Aside from our initial characterizations [65], nuclear actions of the plant-specific RMR1 subfamily remain unknown. Reagents and materials will be synthesized for immunofluorescence detection of functional RMR1 in normal and rmr/mop mutant backgrounds to define both the intracellular and epistatic positions of RMR1 within the nucleus and RdDM/CTGS pathway. These reagents and materials will also be important for basic RMR1 protein characterizations and provide a platform for future biochemical purifications and interaction studies of RMR1 protein partners.

Generate RMR1 Custom Peptide Antibodies

With assistance of peptide chemists at New England Peptides (Gardner, Mass.), four 10-15 amino acid peptides have been identified to optimize high antigenicity, low cross reactivity, and functional relevance. Two peptides flank the conserved C terminal region, and two others flank the RMR1-3 truncation placement in the N-terminal region. We have detected rmr1 mRNA by RT PCR in rmr1-3 homozygotes suggesting that the premature rmr1-3 nonsense codon does not flag the RNA for nonsense-mediated decay. Peptide synthesis, conjugation to keyhole limpet heamocyanin and antisera production will be outsourced to Covance Research Products (AALAC accredited, OLAW #A3850-01, USDA #23-R-0007). Rabbits will be prescreened for general cross reactivity to maize proteins by ELISA, and each conjugated peptide will be injected into two rabbits with low background. Serum will be analyzed by western blot for RMR1 specificity, and antibodies will be purified by either IgG or peptide affinity columns as necessary to reduce non-specific background. Protein extracts from rmr1-3 homozygotes will aid our evaluation and enhancement of RMR1 specificity. These reagents will be useful for both western blot analyses and tissue based in situ hybridizations in addition to their intended use in immunolocalization studies. Similar peptide-based antiseras have been successfully used by Pontes et al. (2006) in these types of studies for PolIVa, PolIVb, DCL3, RDR2, and DRD1.

Generate Functional FLAG-Tagged RMR1 Transgenic Maize

Stable lines expressing a functional 6×FLAG-tagged version of RMR1 will be generated as a backup strategy for immunolocalization studies and to provide an experimental platform for future studies related to RMR1 biochemistry and nuclear actions. Since expression from a constitutive promoter like ubiquitin might lead to non-biologically relevant artifacts in cell or tissue types in which Rmr1 is not typically expressed, we will have the native promoter drive a RMR1-FLAG fusion protein. We will therefore modify a pCAMBIA1300-based Gateway vector [74] to have a 5′ portion of the Rmr1-B73 sequence replace the resident ubiquitin promoter. Non-repetitive 5′ putative promoter sequences will be identified from the recently sequenced BAC CH201-7N19 (AC212361) that we have shown contains the rmr1 locus [65]. We will use PCR and restriction-enzyme based strategies to swap the ubiquitin promoter with as much unique Rmr1-B73 5 sequence as possible. We will design appropriate oligonucleotide primer sets to amplify the protein-coding portion of an rmr1 flcDNA molecule (also see Example 8), introduce these amplicons into a Gateway topoisomerase-based cloning vector [71], and derive both N- and C-terminal FLAG versions with additional modification. These clones will be sequenced and shuttled into our pCAMBIA1300-Rmr1 promoter derivatives for subsequent A. tumefaciens-mediated maize transformation as detailed above in SA1. We will evaluate these 10 lines by genetic segregation tests (locus copy number) and both southern (locus arrangements) and northern (RNA expression) blot hybridizations. To ensure that immunolocalizations represent biologically relevant action, it is essential that the epitope-tagged RMR1 protein is functional. Rapidly dividing tissues of glufosinate-resistant Ti plants will be assayed by western blots for expression of FLAG-RMR1 and positive plants will be crossed by our B73 homozygous T Pl rmr1-1 line. Glufosinate-resistant, FLAG-positive progeny will be crossed by a B73 T Pl rmr1-1/+Pl Rmr1-B73 heterozygote to ensure that all progeny receive a Pl1-Rhoades allele of Pl state. If the RMR1-FLAG constructs genetically complement the rmr1-1 defect, then glufosinate-resistant plants with fully fertile pollen (T/T) and a rmr1-1/rmr1-1 SNP-genotype should have a distinct Pl anther phenotype (FIG. 18). Lines derived from transgenic plants showing rmr1-1 complementation will be used for immunolocalization and epistasis analysis.

Perform Immunolocalization and Epistasis Analyses

Peptide antibodies and/or anti-FLAG antibodies against epitope-tagged RMR1 will be used for in situ detection in maize root-tip cells using epi-fluorescence microscopy. We will also track RMR1 cellular localizations in different mutant backgrounds. For example, RMR1 and MOP1 (ZmRDR2) are both required for the accumulation of siRNAs and likely function together in the RdDM pathway [65]. If RMR1 is mislocalized in mop1 mutants it would suggest that mop1 is epistatic to RMR1. As additional rmr mutants or maize mutants in the RdDM pathway are identified, they will be submitted to similar analyses. This approach, which was successfully employed in Arabidopsis to characterize the RdDM pathway [28], promises to define similar relationships for a novel Snf2 component as well as provide a robust cytological platform for understanding the nuclear dynamics mediating small RNA metabolism and allelic interactions in large grain cereals.

Example 15 Evaluate Effects of RMR1 on Epigenomic Conditioning and Heterosis

To profile the genome-wide effects of RMR1 action and heterosis on small RNA biogenesis, small RNA libraries from inbred rmr1 mutants, wildtype, and F₁ progeny will be characterized using Solexa-based sequencing. Near-isogenic inbred lines will also be tested for heterotic effects of RMR1 function on traits of commercial importance. Based on 1) published data regarding RMR1 effects on Pl1-Rh, 2) the loss of ˜70% of ˜24 nt small RNAs in rmr1 mutants, and 3) our observations related to the breeding behaviors of rmr1 mutants, we hypothesize that different constellations of genomic heterochromatin are conditioned by the presence or absence of RMR function. This differential conditioning of the genome should be reflected in unique small RNA sequence profiles. Given that preconditioning the genome (+/−RMR1 action) appears to give rise to differential developmental responses upon outcrossing (Table 5), unique small RNA sequence profiles are also expected in F₁ epihybrids. To evaluate the dynamics of the RMR1-modulated epigenome, we will compare Solexa-based deep sequencing profiles of small RNAs from rmr1-1/rmr1-1 inbreds (B73), rmr1-1/Rmr1-B73 from marker assisted introgression into B73, and reciprocal rmr1-1/Rmr-B73 and Rmr1-B73/rmr1-1 F₁s. Given preliminary data showing inbred genomes conditioned in the absence of RMR1 function increase progeny dry ear weights nearly 10% in crosses to the B73 inbred (Table 5), a more rigorous test of the epigenetic complementation concept is also highly motivated. These experiments will address a hypothesis regarding the role of epigenetic complementation in heterosis and provide defining information regarding RMR1 action in the maize genome.

Develop Germplasm Resources

The A619, A632, B73, and Mol7 inbred lines were obtained directly from the North Central Plant Introduction Station (Ames, Iowa). Prior to molecular identification, rmr1 mutant alleles had been committed to backcross programs requiring concomitant introgression of a reporter Pl1-Rhoades allele linked to the T6-9 (043-1) breakpoint. Molecular SNP-based genotyping will rapidly advance these introgressions in the absence of the translocation chromosome so that the A619/A632 materials can be combined. Regardless of the experimental outcomes, these efforts will provide introgressed rmr1 mutant lines that will be deposited to the Maize Genetics Cooperative Stock Center for curation and public dissemination.

Perform Epihybrid Crosses and Prepare Small RNA Libraries

Using B73 as recurrent pistillate parent, rmr1-1 has been introgressed and subsequently recovered in homozygous condition. The rmr1-1 ^ B73 line (˜97% B73) will be advanced to the S2 generation prior to analysis. As Pl reversion tests in rmr1 mutants suggest a parent of origin effect [10], it is desirable to sample rmr1⁻ conditioned genomes through both pistil and pollen transmission. Reciprocal crosses will be made between rmr1-1/rmr1-1 and Rmr1-B73/rmr1-1 parents. Next, congenic parents will be grown alongside reciprocal F₁ epihybrids and developing ears (5 cm) will be collected for RNA isolations. The >50 g of enriched small RNA we routinely isolate from individual ears will provide more than enough material to assemble independent small RNA libraries. We will use the reagent package from illumina to clone libraries for Solexa sequencing using fractionated and PAGE purified small RNAs according to Illumina protocols. We will make 8 individual libraries (2 biological replicates) for a single Solexa flow-cell sequencing run.

Conduct Small RNA Profiling and Downstream Bioinformatics

The University of California at Berkeley operates a Functional Genomics Laboratory (FGL) for full support of Solexa sequencing projects. We will provide FGL with the prepared libraries, a 1 Tb data storage device and the most up-to-date B73 maize genome sequence to obtain alignment data for export and evaluation. Downstream analysis of the reads and alignments will be guided by prior 454 sequencing analyses carried out on rdr2 mutants [87,60] It is expected that, as was seen in the rdr2 analyses, there will be clear trends in the quantity and types of RMR1-dependent RNA sequences in addition to the identification of all the maize microRNAs and trans-acting siRNAs. Although full analytical power of these profiles awaits the final maize genome assembly we will use sliding-window alignment profiles [60] in available assemblies of high confidence to evaluate position-dependent representations. Thus both in the short and long-term, this data set promises to highlight specific dynamic changes to the maize epigenome represented by RMR1-dependent conditioning.

Evaluate Heterotic Traits in Conditioned Epigenotypes

Respective combinations of inbred lines B73 or A632 (Reid Yellow Dent) and Mol7 or A619 (Lancaster Sure Crop) represent one of the most highly utilized heterotic patterns in the commercial hybrid industry. For each BC4F2 progeny, plants homozygous for either inbred Rmr1 or rmr1 mutant alleles will be identified by SNP-typing and reciprocally crossed with plants from the inbred heterotic parent (i.e. rmr1-1 ^ A619 A632; A632 rmr1-1 ^ A619; A619 rmr1-1 ^ A632; rmr1-1 ^ A632 A619). Five plots will be evaluated from each heterotic cross for days-to-flowering, plant height at anthesis, and dry kernel weights. For each plot, three 20-kernel rows will be planted side by side and only plants of the interior rows will be sampled. Each of the test plots will be assigned randomized field placements to effectively neutralize environmental variations and will all be planted on the same day. Plants will be open pollinated and individual ears will be hand-harvested, dried in a 100 F room briefly to minimize kernel damage and shelled for weight measurements. Kernels will be further dried to 12-15% moisture content and the kernels per plant will be directly weighed. Test weights (lbs/bushel) per plot will be approximated using a hand-held balance. Grain yield estimates of Mg per hectare will be calculated per plot and per genotype. Mean values+/−s.e.m. for each set of 5 test plots will be compared using a two-tailed z-test. Should these initial studies with A619 and A632 reveal biomass and/or grain yield increases, this experimental design will be repeated with rmr1 mutations introgressed to ˜99%, and we will forge collaborations for field-based yield trials. If no differences are indicated, we will still replicate the experimental design with B73 and Mol7 combinations. As it is possible that epigenome conditioning is progressive and cumulative, the experimental design will be repeated using appropriate BC4F2S3 A619 and A632 lines. Given the pedigree of these materials, it is expected that provenance tests, measurements of these or additional traits, and statistical treatments can be used to make estimates of epigenetic variation contributions to broad-sense heritability.

TABLE 1 Testcross results measuring establishment of Pl′ in T Pl-Rh rmr1-2/+ Pl′ rmr1-1 plants Progeny No. of individual progeny Pl-Rh structural with specific anther color scores Parental cross tester genotype 1 2 3 4 5 6 7 05-542-6 5 05-103-1 A632 +/+ 0 3 0 0 0 0 0 05-103-1 5 05-542-6 A632 +/+ 1 6 0 2 0 0 0 06-530 X 06-316-10 A632 +/+ 0 2 3 0 0 0 0 05-539-1 5 05-542-6 A619 +/+ 1 2 0 1 0 0 5 05-539-2 5 05-541-6 A619 +/+ 0 0 1 0 1 3 8 05-541-6 5 05-539-2 A619 +/+ 0 3 2 4 1 1 0 06-528 X 06-316-12 A619 +/+ 0 0 0 3 2 5 4 05-528 X 06-316-10 A619 +/+ 0 0 0 0 0 0 11 05-528 X 06-316-10 A619 +/+ 0 0 0 0 0 1 7 05-542-6 5 05-103-1 A632 T/+ 0 4 5 3 0 0 0 05-103-1 5 05-542-6 A632 +/T 0 5 0 2 0 0 1 06-530 X 06-316-10 A632 +/T 1 11 1 0 0 0 0 05-539-1 5 05-542-6 A619 +/T 0 0 1 0 1 0 5 05-539-2 5 05-541-6 A619 +/T 0 0 0 1 0 0 2 05-541-6 5 05-539-2 A619 T/+ 0 1 2 1 2 0 0 06-528 X 06-316-12 A619 +/T 0 4 2 1 0 0 0 05-528 X 06-316-10 A619 +/T 0 0 0 0 0 0 4 05-528 X 06-316-10 A619 +/T 0 0 0 0 0 0 5 Totals: +/+ 2 16 6 10 4 10 35 T/+ 0 5 7 4 2 0 0 +/T 1 20 4 4 1 0 17

TABLE 2 Evaluation of b1 paramutation in rmr1/rmr1; B-I/B′ plants through crosses to Rmr1 b1 testers No. of progeny with specific plant Staminate Parental plant Allele phenotypes parent phenotype tested B-I B′ 02-508-22 Dark B-I rmr1-1 0 60 02-508-60 B-I rmr1-1 4 30 02-514-8 Dark B′ rmr1-3 0 53 02-514-73 B-I rmr1-3 0 59 Totals 4 202

TABLE 3 Complementation tests Progeny No. of individuals with specific Parents No. of anther color scores Pistillate Staminate ears 1 2 3 4 5 6 7 mop1-3 (ems98262) +/mop1-1 ems98262/ems98262 1 2 8 2 0 0 0 3 ems98262/ems98262 +/mop1-1 1 6 0 3 0 0 0 5 +/ems98262 rmr1-1/rmr1-1 2 4 19 17 0 0 0 0 +/ems98262 rmr2-1/rmr2-1 1 2 14 10 0 0 0 0 +/ems98262 rmr6-1/rmr6-1 1 0 6 17 2 0 0 0 +/rmr6-1 ems98262/ems98262 1 6 10 7 1 0 0 0 mop1-4 (ems98941) +/mop1-1 ems98941/ems98941 2 5 1 1 0 0 0 12 +/mop1-3 ems98941/ems98941 2 1 14 1 2 0 0 8 rmr1-1/rmr1-1 ems98941/ems98941 2 1 12 1 1 0 0 0 rmr2-1/rmr2-1 ems98941/ems98941 2 10 11 2 4 0 0 0 +/rmr6-1 ems98941/ems98941 2 7 15 2 1 0 0 0 rmr1-3 (ems98287) +/mop1-3 ems98287/ems98287 1 10 7 4 0 0 0 0 ems98287/ems98287 +/rmr1-1 2 11 16 2 0 0 0 20 +/ems98287 rmr2-1/rmr2-1 1 13 13 0 0 0 0 0 ems98287/ems98287 rmr6-1/rmr6-1 1 18 3 0 0 0 0 0 rmr1-4 (ems051069) +/mop1-4 ems051069/ems051069 1 7 13 2 3 0 0 0 +/rmr1-1 ems051069/ems051069 1 0 7 6 1 0 0 13 +/rmr1-3 ems051069/ems051069 1 3 6 2 0 0 0 15 +/rmr2-1 ems051069/ems051069 1 8 17 4 0 0 0 0 +/rmr6-1 ems051069/ems051069 1 2 13 12 0 0 0 0

TABLE 4 PCR oligonucleotide primers used Primer Name Sequence (5′ -> 3′) SEQ ID NO Os10F GAA GAG TTG GGT GTG GGA AA 30 Os10R AAC ATC TTG CTC TGG CCT GT 31 ExonF GTG TCA GTG TTT GCC GAG AA 32 ExonR CTA TAG TTA CAA TCA AAT TGC TAC TGA G 33 mid5′F GTT GCA GCA ACA GAA TCT AGC 34 Os11F AAG CAT ACA TCG CCC AAA TC 35 Os15R CAC GCT GAT GCT CAT ACA TA 36 Os12F CCC AAT CAT GCA ACT CCT CT 37 Os13Rb CCA TAA CTT ATT CCA AGA ATA CC 38 IntronF GTC GTT TCC CAG AAT AGA GTA ACT TTG 39 IntronR GAT ATG CAG AAT ACT ACA TAT GTT TGA G 40 CAPS6F GCA TCT TCG CAA GTT CTT CA 41 CAPS6R TCG TGG GAA GTC ATC TCC TC 42 CAPS10F ATG AGT AGT GCC CCA TCC AG 43 CAPS10R TCA GCC TCT TCT TCC TCT TCC 44 Intron0 F2 GAT GAC GCT TCT CAT GAC G 45 Intron0 R2 ATC ATC GTA GTC ATC TTC AAA GG 46

TABLE 5 Trait analysis of progeny derived from single pollen sources of different rmri genotypes Statistical Pistillate Staminate inbred genotype^(b) significance^(c) inbred Trait^(a) Rmr1-A632/Rmr1-A632 rmr1-1/rmr1-1 z P A632 ACS 1.7 ± 0.1 (14) 5.2 ± 0.4 (20) 8.8 <<0.01 Height 223.2 ± 1.2 (14) 221.1 ± 1.2 (20) 1.2 n.s. DTF 59.4 ± 0.3 (14) 57.1 ± 0.1 (20) 6.3 <<0.01 Ear weight 87.9 ± 3.6 (8) 85.2 ± 1.6 (13) 0.7 n.s. Mo17 ACS 1.8 ± 0.3 (19) 4.9 ± 0.2 (16) 8.5 <<0.01 Height 246.2 ± 0.8 (19) 238.8 ± 2.2 (16) 0.4 <0.01 DTF 57.6 ± 0.2 (19) 57.2 ± 0.3 (16) 1.1 n.s. Ear weight 122.2 ± 3.4 (12) 123.8 ± 3.6 (8) 0.3 n.s. B73 ACS 1.9 ± 0.1 (18) 4.8 ± 0.3 (16) 9.1 <<0.01 Height 239.1 ± 1.9 (18) 213.4 ± 9.6 (19) 5.4 <<0.01 DTF 59.8 ± 0.2 (18) 58.4 ± 0.3 (17) 3.8 <<0.01 Ear weight 91.8 ± 3.0 (12) 101.3 ± 1.8 (11) 2.6 <0.01 F₂S₁ plants homozygous for either Rmr1-A632/Rmr1-A632 or rmr1-1/rmr1-1 were identified and used to generate lines through single seed descent. ^(a)Anther Color Scores, (ACS; [7]), plant height in cm at anthesis (height), days to flowering or first pollen shed (DTF), and dry ear weight in grams from open pollinations were measured on individual progeny. ^(b)Means ± s.e.m. with number of individual progeny measured in parentheses. ^(c)A two-sample z-test was applied to the null hypothesis that the difference in mean values between the two progeny sets is due to random chance. n.s. indicates a non-significant P value of >0.05.

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What is claimed is:
 1. A method for reducing or mitigating gene silencing of a transgene, comprising: providing a transgenic plant with a transgene; and introducing a recombinant nucleic acid to said plant, wherein said recombinant nucleic acid comprises a nucleic acid in an antisense orientation wherein the sense orientation of said nucleic acid encodes the polypeptide of SEQ ID NO: 1, wherein said antisense nucleic acid reduces expression of the encoded SEQ ID NO: 1 polypeptide, and wherein the level of expression of said transgene is increased relative to the level of expression of said transgene in a transgenic plant that does not contain said recombinant nucleic acid.
 2. A method for reducing or mitigating gene silencing of a transgene, comprising: providing a transgenic plant with a transgene; and introducing a recombinant nucleic acid to said plant, wherein said recombinant nucleic acid comprises an RNA interference (RNAi) construct comprising at least a fragment of 20 contiguous nucleotides of a nucleic acid that encodes the polypeptide of SEQ ID NO: 1, wherein said RNAi construct reduces expression of the encoded SEQ ID NO: 1 polypeptide, and wherein the level of expression of said transgene is increased relative to the level of expression of said transgene in a transgenic plant that does not contain said recombinant nucleic acid.
 3. A transgenic plant comprising a recombinant nucleic acid including a nucleic acid in an antisense orientation wherein the sense orientation of said nucleic acid encodes the polypeptide of SEQ ID NO: 1, and wherein said antisense nucleic acid reduces expression of the encoded SEQ ID NO: 1 polypeptide.
 4. A transgenic plant comprising a recombinant nucleic acid including an RNA interference (RNAi) construct comprising at least a fragment of 20 contiguous nucleotides of a nucleic acid that encodes the polypeptide of SEQ ID NO: 1, wherein said RNAi construct reduces expression of the encoded SEQ ID NO: 1 polypeptide.
 5. Seed from the transgenic plant of claim 3, wherein the seed comprises a recombinant nucleic acid including a nucleic acid in an antisense orientation wherein the sense orientation of said nucleic acid encodes the polypeptide of SEQ ID NO:
 1. 6. Seed from the transgenic plant of claim 4, wherein the seed comprises a recombinant nucleic acid including an RNAi construct comprising at least a fragment of 20 contiguous nucleotides of a nucleic acid that encodes the polypeptide of SEQ ID NO:
 1. 